WO2018235938A1

WO2018235938A1 - Methods for sequencing and analyzing nucleic acids

Info

Publication number: WO2018235938A1
Application number: PCT/JP2018/023778
Authority: WO
Inventors: 克之城口
Original assignee: 国立研究開発法人理化学研究所
Priority date: 2017-06-23
Filing date: 2018-06-22
Publication date: 2018-12-27
Also published as: JP7160349B2; JPWO2018235938A1

Abstract

The present invention provides methods for correcting errors which occur in the digital assay of nucleic acids using molecular bar codes. More specifically, provided are: a method for identifying mispairing of molecular bar codes and index sequences in accordance with the detection frequency; a method for identifying molecular bar codes having base substitutions which have been categorized into the same cluster; and a method for identifying molecular bar codes having insertions or deletions using molecular bar codes including fixed bases and random bases.

Description

Method of sequencing and analyzing nucleic acid

Reference to related applications

This application is an application benefiting from the priority of US Provisional Application No. 62 / 523,857 (filing date: June 23, 2017), which is incorporated herein by reference in its entirety. It shall be.

The present invention relates to methods of sequencing and analyzing nucleic acids.

The development of next-generation sequencer platforms has made it possible to simultaneously analyze the sequences of a large number of nucleic acids in one run. By adding a unique molecular barcode to each molecule of nucleic acid molecules present in the sample, the number of unique molecular barcode types can be made to correspond to the number of nucleic acid molecules, and the platform of the next generation sequencer The way of digital determination of H. pylori was pioneered (Patent Document 1 and Non-Patent Document 1). By using a molecular barcode as a random base and lengthening the base sequence, it becomes possible to easily add great diversity to the barcode sequence, and the dynamic range of the nucleic acid molecule that can be quantified digitally is expanded (Patent Document 1 and Non Patent Literature 1).

However, in digital quantification, the sequence of molecular barcodes may change during analysis, whereby newly generated molecular barcodes may affect the accuracy of quantification of nucleic acid molecules. However, if the sequence of the molecular barcode is randomly designed, it is difficult to grasp that the sequence has changed. In addition, due to the randomness of the molecular barcode sequence, it is difficult to analyze what kind of error may occur in digital quantification, and it is also difficult to present the solution.

U.S. Patent No. 9,260,753

The present invention provides methods of sequencing and analyzing nucleic acids.

The present inventors use the index and the barcode in the digital quantification method of the target nucleic acid molecule, when mixing a plurality of samples and quantifying the target nucleic acid molecule, the nucleic acid derived from the sample whose index is different from unexpected It is clarified that a miss index can occur, which is added to We also consider the most frequent pair to be the correct pair when two different indexes are added to the same barcode, and excluding any or all others as miss indexes. , Revealed that the accuracy of the digital quantification method can be improved.
We have found that when counting the number of types of barcode sequences, mutations (e.g. insertions, substitutions and deletions) occur in the barcode sequence and the sequences to be judged to be the same are different sequences It has become clear that perceived problems can occur. The present inventors have clarified that the accuracy of the digital quantification method can be improved by clustering sequences having a certain sequence similarity into a group and quantifying a target nucleic acid molecule based on the number of clusters.
We have found that when digitally counting nucleic acids, the problem of misidentifying the template can occur. We also identify the most frequent pair as the correct pair and exclude any or all others as false identifications when two different target nucleic acid sequences are added to the same barcode It was revealed that the accuracy of the digital quantification method could be improved.

That is, according to the present invention, the following inventions are provided.
(1A) A method of analyzing nucleic acid:
(I) subjecting a mixture of a plurality of target nucleic acid molecules to which a molecular barcode and an index have been added to sequencing to obtain sequence information;
(II) From the sequence information obtained in (I) above, a sequence having a specific index or a sequence similar thereto, and / or a sequence having a specific molecular barcode or a sequence analogous thereto is selected and selected Creating a group by the arranged array;
(III) determining, in the group created in (II) above, the pair of the index with the highest detection frequency and the molecular barcode as the correct pair of the index and the molecular barcode;
Method, including.
(2A) The method according to (1A) above, wherein the target nucleic acid molecule to which at least a molecular barcode has been added is subjected to amplification prior to step (I).
(3A) A sequence similar to the sequence having a specific molecular barcode in step (II) is a sequence including the sequence having the specific molecular barcode and a mismatched base having a number of bases or less in the molecular barcode sequence portion. The method according to (1A) or (2A) above.
(4A) The method according to any of the above (1A) to (3A), wherein the molecular barcode has a fixed base at a specific position.
(5A) that the sequence similar to the sequence having the specific molecular barcode in step (II) contains the fixed base at the specific position, and / or the position of the fixed base is shifted from the specific position The method according to (4A) above, which is selected based on
(6A) The method according to (4A) above, further comprising excluding from analysis the sequence having a molecular barcode that does not contain the fixed base at the specific position.
(7A) In step (III), excluding the index and molecular barcode pairs other than the determined correct pairs as the index and molecular barcode mispairing,
The method according to any one of the above (1A) to (5A).
(8A) further comprising the step of determining the number of target nucleic acid molecules contained in the sample from which the target nucleic acid molecule is derived based on the number of groups having a specific molecular barcode or a sequence similar thereto And the method according to any one of (1A) to (7A) above.
(9A) A method of analyzing nucleic acid:
(I) subjecting a mixture of a plurality of nucleic acid molecules to which molecular barcodes have been added to sequencing to obtain sequence information;
(II) selecting a sequence having a specific molecular barcode or a sequence similar thereto from the sequence information obtained in (I) above, and creating a group from the selected sequence;
Method, including.
(10A) A sequence similar to the sequence having the specific molecular barcode in step (II) is a sequence including the sequence having the specific molecular barcode and a mismatched base having a number of bases or less in the molecular barcode sequence portion. The method according to (9A) above.
(11A) The method according to (9A) or (10A) above, wherein the molecular barcode has a fixed base at a specific position.
(12A) that the sequence similar to the sequence having the specific molecular barcode in step (II) contains the fixed base at the specific position, and / or the position of the fixed base is shifted from the specific position The method according to (11A) above, which is selected based on
(13A) The method according to (11A), further including the step of excluding from analysis the sequence having a molecular barcode which does not contain the fixed base at the specific position.
(14A) further comprising the step of determining the number of target nucleic acid molecules contained in the sample from which the target nucleic acid molecule is derived based on the number of groups having a specific molecular barcode or a sequence similar thereto And the method according to any one of (9A) to (13A) above.
(15A) The method according to any one of (9A) to (14A) above, wherein the target nucleic acid molecule to which at least a molecular barcode has been added is subjected to amplification prior to step (I).
(16A) A method of analyzing nucleic acid:
(I) subjecting a mixture of a plurality of nucleic acid molecules to which a molecular barcode having a fixed base at a specific position is added to sequencing to obtain sequence information;
(IIa) excluding from analysis a sequence having a molecular barcode which does not contain the fixed base at the specific position;
(IIb) obtaining sequence information comprising a sequence comprising the fixed base at the specific position in the step (I) or after the step (I); or (IIc) as the step (II) above Further comprising the step of selecting a sequence having a specific molecular barcode or a sequence similar thereto from the sequence information obtained in 4.), and creating a group according to the selected sequence, and in step (II) or After II) obtaining a group consisting of a sequence comprising the fixed base at the particular position;
Method, including.

According to the present invention, the following inventions are also provided.
(1B) obtained by sequencing using a mixture of a plurality of samples containing a target nucleic acid molecule having an index unique to each sample containing a plurality of nucleic acid molecules and a unique or arbitrary molecular barcode unique to each nucleic acid molecule A method for determining the correct pair or mispair of an index added to a target nucleic acid molecule and a molecular barcode from sequence information.
(E) From the obtained sequence information, a sequence having a specific index or a sequence similar thereto, a sequence having a specific molecular barcode or a sequence similar thereto, or a sequence containing a target nucleic acid molecule or a similar Selecting a sequence and creating a group from the selected sequence;
(F) In the group created in (E) above, determine the pair with the highest detection frequency index and molecular barcode as the correct pair of index and molecular barcode, and / or the low detection frequency index and molecules Determining at least any one or all of the barcode pairs as an index and a molecule barcode mispairing;
Method, including.
(2B) In step (E), select an array having a specific index and create a group for each index,
In step (F), for nucleic acid sequences having molecular barcodes that appeared in a plurality of groups, the barcode / index pair with the highest number of reads is determined as the correct barcode / index pair, or Determine the highest index and molecular barcode pair as the correct index and molecular barcode pair
The method according to (1B) above.
(3B) In step (E), select a sequence having a specific molecular barcode to create a group for each molecular barcode,
In the step (F), the highest detection frequency index and molecular bar code pair in the created group is determined as the correct index and molecular bar code pair.
The method according to (1B) above.
(4B) In step (E), select a sequence containing the sequence of the target nucleic acid molecule to create a group,
In step (F), a sequence having a specific index is further selected from the group to create a subgroup, and a barcode having the largest number of reads and a nucleic acid sequence having a molecular barcode appearing in a plurality of subgroups Determine the index pair as the correct barcode / index pair, or determine the index / molecular barcode pair with the highest detection frequency as the correct index / molecular barcode pair,
The method according to (1B) above.
(5B) In step (E), select a sequence containing the sequence of the target nucleic acid molecule to create a group,
In step (F), molecules having a specific molecular barcode are further selected from the group to create a subgroup, and the index with the highest detection frequency and molecular barcode in one created subgroup is indexed And determine the correct pair of molecular barcodes,
The method according to (1B) above.
(6B) In the step (F), at least any one or all of the index and molecular barcode pairs other than the determined correct pair are determined as the index and the molecular barcode mispairing,
The method according to (2B) to (5B) above.
(7B) select a molecule having a specific index in step (E) and create a group for each index,
In the step (F), with respect to the sequences having molecular barcodes appearing in a plurality of groups, a low detection frequency index and molecular barcode pair is determined as at least any one or all of the indexes and the molecular barcode mispair ,
The method according to (1B) above.
(8B) select a sequence having a specific molecular barcode in step (E) to create a group for each molecular barcode,
In the group created in the step (F), the pair of the low detection frequency index and the molecular barcode is determined as the index and / or the molecular barcode, at least any one or all of mispairs,
The method according to (1B) above.
(9B) selecting a sequence containing the target nucleic acid molecule in step (E) to create a group,
In step (F), molecules having a specific index are further selected from the group to create subgroups, and for nucleic acid molecules having molecular barcodes that appear in a plurality of subgroups, an index with low detection frequency and a molecular barcode Determine at least any one or all of the pairs as the index and the molecule barcode mispairing,
The method according to (1B) above.
(10B) selecting a molecule containing the target nucleic acid molecule in step (E) to create a group,
In step (F), a molecule having a specific molecular barcode is selected from the group to create a subgroup, and at least one of a pair of a low detection index and a molecular barcode in one created subgroup Determine one or all of the index and molecular barcodes as a spare
The method according to (1B) above.
(11B) In step (E), the step of creating a group creates a group by clustering, as a group, molecules assumed to have the same sequence determined based on sequence identity or similarity Done by
The method according to (1B) to (10B) above.
(12B) In step (E), clustering is
(I) In the sequence of the molecular barcode portion, by grouping nucleic acid molecule groups having the same sequence as the unique molecular barcode sequence into the same cluster;
(Ii) in the sequence of the molecular barcode portion, by grouping nucleic acid molecule groups having a unique molecular barcode sequence and a sequence having up to one mismatch into the same cluster;
(Iii) in the sequence of the molecular barcode portion, by grouping nucleic acid molecules having a unique molecular barcode sequence and a sequence having up to 2 bases of mismatch into the same cluster; or (iv) the molecular barcode In the partial sequence, it is performed by classifying nucleic acid molecule groups having a sequence having a unique molecular barcode and a mismatch up to 3 bases into the same cluster,
The method according to (11B) above.
(13B) In step (E), clustering is
In the sequence of the molecular barcode portion, by grouping nucleic acid molecule groups having a sequence sequenced as having an insertion or deletion (indel) of bases into the same cluster,
The method according to (11B) or (12B) above.
(14B) In step (E), clustering is
In the sequence of the molecular barcode portion, it is performed on a group of nucleic acid molecules obtained excluding the sequence sequenced as having a base insertion or deletion (indel),
The method according to (11B) or (12B) above.
(15B) The insertions or deletions of the bases in the position of each of the one or more fixed bases arranged in all molecular barcode sequences linked to the nucleic acid molecule and in the sequence of the sequenced molecular barcode sequence part The method according to (13B) or (14B) above, further comprising specifying by position differences with the position of each of the one or more fixed bases.
(16B) obtained by sequencing using a mixture of a plurality of samples containing a target nucleic acid molecule having a unique index for each sample containing a plurality of nucleic acid molecules and a specific or arbitrary molecular barcode attached to each nucleic acid molecule A method of determining the number of target nucleic acid molecules contained in a specific original sample from sequence information,
(E) selecting a nucleic acid molecule containing the sequence of the target nucleic acid molecule from the obtained sequence information;
(F) clustering the nucleic acid molecules selected in (e) above for each unique molecular barcode sequence, and then identifying a cluster having a plurality of sequences in the index nucleic acid molecule portion,
(G) In each of the clusters identified in (f), the index with the highest detection frequency and the molecular barcode pair are identified as the correctly indexed target nucleic acid molecules, and the other index and molecular barcode pairs are identified. Determining that it is a mispair, and
Including
The target nucleic acid molecule contained in the sample corresponding to the index, the number of kinds of sequence of the unique molecular barcode linked to the target nucleic acid molecule correctly indexed (or the number of clusters of the target nucleic acid molecule correctly indexed) Is the number of
Method.
(17B) In (f), clustering is
(I) In the sequence of the molecular barcode portion, by grouping nucleic acid molecule groups having the same sequence as the unique molecular barcode sequence into the same cluster;
(Ii) in the sequence of the molecular barcode portion, by grouping nucleic acid molecule groups having a unique molecular barcode sequence and a sequence having up to one mismatch into the same cluster;
(Iii) in the sequence of the molecular barcode portion, by grouping nucleic acid molecules having a unique molecular barcode sequence and a sequence having up to 2 bases of mismatch into the same cluster; or (iv) the molecular barcode In the partial sequence, it is performed by classifying nucleic acid molecule groups having a sequence having a unique molecular barcode and a mismatch up to 3 bases into the same cluster,
The method as described in the above (16B).
(18B) In (e), clustering is
In the sequence of the molecular barcode portion, by grouping nucleic acid molecule groups having a sequence sequenced as having an insertion or deletion (indel) of bases into the same cluster,
The method according to (16B) or (17B) above.
(19B) In (e), clustering is
In the sequence of the molecular barcode portion, it is performed on a group of nucleic acid molecules obtained excluding the sequence sequenced as having a base insertion or deletion (indel),
The method according to (16B) or (17B) above.
(20B) The insertions or deletions of the bases in the position of each of the one or more fixed bases arranged in all the molecular barcode sequences linked to the nucleic acid molecule and in the sequence of the sequenced molecular barcode sequence part The method according to (18B) or (19B) above, further comprising specifying by difference with the position of each of the one or more fixed bases.
(21B) In the digital quantification of a target nucleic acid molecule using a barcode sequence, based on the information on the obtained nucleic acid sequence, the sequence of the molecule barcode after mutation has one group together with other sequences having sequence similarity And clustering the number of target nucleic acid molecules based on the number of clusters obtained.
(22B) The method according to (21B) above, wherein clustering is
(I) In the sequence of the molecular barcode portion, by grouping nucleic acid molecule groups having the same sequence as the unique molecular barcode sequence into the same cluster;
(Ii) in the sequence of the molecular barcode portion, by grouping nucleic acid molecule groups having a unique molecular barcode sequence and a sequence having up to one mismatch into the same cluster;
(Iii) in the sequence of the molecular barcode portion, by grouping nucleic acid molecules having a unique molecular barcode sequence and a sequence having up to 2 bases of mismatch into the same cluster; or (iv) the molecular barcode A method carried out by classifying nucleic acid molecule groups having sequences having a unique molecular barcode and up to 3 bases of mismatches into the same cluster in the partial sequence.
(23B) The method according to (21B) or (22B) above, wherein the clustering is insertion of bases (eg, up to 1 base, up to 2 bases, or up to 3 bases) in the sequence of the molecular barcode portion A method carried out by classifying nucleic acid molecule groups having sequences sequenced as having a deletion (indel) into the same cluster.
(24B) The method according to any one of (21B) to (23B) above,
The relative position of each of the one or more fixed bases located in all of the molecular barcode sequences linked to the nucleic acid molecule and the position of each of the one or more fixed bases in the sequence of the sequenced molecular barcode sequence portion Detection of base insertions or deletions (indels) by identification by comparing the
Clustering is performed by grouping nucleic acid molecule groups having sequences sequenced as having base insertions or deletions (indels) in the sequence of the molecular barcode portion into the same cluster, or
A method wherein clustering is performed on a group of nucleic acid molecules obtained by excluding sequences sequenced as having a base insertion or deletion (indel) in the sequence of the molecular barcode portion.
(25B) A method for detecting insertion or deletion (indel) of a base in a barcode in digital quantification of a target nucleic acid molecule using the barcode sequence, wherein all molecular barcode sequences linked to the nucleic acid molecule By comparing the relative position of the position of each of the one or more fixed bases located in the and the position of each of the one or more fixed bases in the sequence of the sequenced molecular barcode portion. A method comprising detecting a base insertion or deletion (indel).

According to the present invention, the following inventions are also provided.
(1C) A unique index for each sample containing a plurality of nucleic acid molecules (meaning index sequence nucleic acid molecule, which may contain a plurality of index nucleic acid molecules if unique to each sample) and unique to each nucleic acid molecule Obtained from sequencing (ie, multiplex sequencing) using a mixture of multiple samples containing the target nucleic acid molecule (eg, DNA or RNA) to which or any molecular barcode (barcode sequence nucleic acid molecule) has been added A method for determining the correct pair or mispair of an index added to a target nucleic acid molecule and a molecular barcode from the sequence information obtained,
(A) separately obtaining a plurality of samples containing a nucleic acid molecule (eg, DNA or RNA) and {at least one of the samples includes a target nucleic acid molecule},
(B) {e.g., in each of a plurality of obtained samples} before amplification of the nucleic acid molecules contained in the sample, each nucleic acid molecule of interest is linked to each of the nucleic acid molecules, or each molecular barcode unique to each nucleic acid molecule is linked; Obtaining a target nucleic acid molecule to which different molecular barcodes are linked;
(C) {For example, before mixing a plurality of samples} A target nucleic acid molecule in which a unique index is added to the target nucleic acid molecule for each sample containing a plurality of target nucleic acid molecules, and different indexes are linked for each sample derived (Step C may be performed after step B, and step B may be performed after step C; and nucleic acid molecules are amplified after step B or C to obtain a target nucleic acid molecule) Amplification products can be obtained),
(D) in a mixture containing the amplification products of the nucleic acid molecule obtained after (B) and (C) above (the sample is mixed after step (C) and after the sample is mixed (B) ) May be performed, or all samples may be mixed after performing step (B), and it is after step (B) that the amplification product of the nucleic acid molecule to which the molecular barcode is linked is obtained. The sample may be mixed before obtaining the amplification product, and after obtaining the amplification product, the sample containing the amplification product may be mixed), an index unique to each sample, and a unique or optional for each target nucleic acid molecule And sequencing the nucleic acid molecule to which the molecular barcode of S is added to determine the sequence of the index portion, the sequence of the molecular barcode portion, and the sequence of the target nucleic acid molecule portion linked thereto as needed for each nucleic acid molecule. When,
(E) From the obtained sequence information {eg, which can be done based on sequence identity or similarity} sequence with a specific index or a sequence similar thereto, sequence with a specific molecular barcode or Selecting a sequence similar to this, or a sequence containing a target nucleic acid molecule or a sequence similar thereto, and creating a group according to the selected sequence;
(F) In the group created in (E) above, determine the pair with the highest detection frequency index and molecular barcode as the correct pair of index and molecular barcode, and / or the low detection frequency index and molecules Bar code pair (for example, a detection frequency pair lower than a predetermined reference value, and the predetermined reference value is 99.5% or less, 99% or less, 90% or less, 80% or less, 70% or less, 60% or less) %, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 1% or less, but is not limited thereto. Determining at least any one or all of the following two) as an index and a molecule barcode mispair:
Method, including.
(2C) In step (E), select an array having a specific index and create a group for each index,
In step (F), for nucleic acid sequences having molecular barcodes that appeared in a plurality of groups, the barcode / index pair with the highest number of reads is determined as the correct barcode / index pair, or Determine the highest index and molecular barcode pair as the correct index and molecular barcode pair
The method according to (1C) above.
(3C) In step (E), select a sequence having a specific molecular barcode and create a group for each molecular barcode,
In the step (F), the highest detection frequency index and molecular bar code pair in the created group is determined as the correct index and molecular bar code pair.
The method according to (1C) above.
(4C) In step (E), select a sequence containing the sequence of the target nucleic acid molecule to create a group,
In step (F), a sequence having a specific index is further selected from the group to create a subgroup, and a barcode having the largest number of reads and a nucleic acid sequence having a molecular barcode appearing in a plurality of subgroups Determine the index pair as the correct barcode / index pair, or determine the index / molecular barcode pair with the highest detection frequency as the correct index / molecular barcode pair,
The method according to (1C) above.
(5C) In step (E), select a sequence containing the sequence of the target nucleic acid molecule to create a group,
In step (F), molecules having a specific molecular barcode are further selected from the group to create a subgroup, and the index with the highest detection frequency and molecular barcode in one created subgroup is indexed And determine the correct pair of molecular barcodes,
The method according to (1C) above.
(6C) In step (F), at least any one or all of the index and molecular barcode pairs other than the determined correct pair are determined as the index and molecular barcode mispairing
The method according to any one of the above (2C) to (5C).
(7C) In step (E), select a molecule having a specific index and create a group for each index,
In the step (F), with respect to the sequences having molecular barcodes appearing in a plurality of groups, a low detection frequency index and molecular barcode pair (for example, a detection frequency pair lower than a predetermined reference value and constant) The reference value includes, but is not limited to, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 1% or less in the group. (A pair of detection frequency pairs) may be determined as a molecule barcode mispair with at least any one or all of the indices.
The method according to (1C) above.
(8C) select a sequence having a specific molecular barcode in step (E) to create a group for each molecular barcode,
Among the groups created in step (F), a pair with a low detection frequency index and a molecular barcode (for example, a pair with a detection frequency lower than a certain reference value, and a certain reference value is 50% or less in the group 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 1% or less, and is not limited thereto, and, for example, even the second and subsequent detection frequency pairs ) Is determined as a mispair of at least one or all of the index and molecular barcodes,
The method according to (1C) above.
(9C) selecting a sequence containing the target nucleic acid molecule in step (E) to create a group,
In step (F), molecules having a specific index are further selected from the group to create subgroups, and for nucleic acid molecules having molecular barcodes that appear in a plurality of subgroups, an index with low detection frequency and a molecular barcode (Eg, a detection frequency pair lower than a predetermined reference value, and the predetermined reference value is 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less) And a value of 1% or less, but is not limited thereto, and may be, for example, a pair of second or later detection frequency) and at least any one or all of index and mispair of molecular barcode decide,
The method according to (1C) above.
(10C) In step (E), select a molecule containing a target nucleic acid molecule to create a group,
In step (F), molecules having specific molecular barcodes are further selected from the group to create subgroups, and a pair of infrequently detected index and molecular barcodes in one created subgroup (eg, constant) The detection frequency is lower than the standard value of the group, and the fixed standard value is 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 1% or less in the group In addition, for example, at least any one or all of the second and subsequent detection frequency pairs may be determined as the index and the molecular barcode mispair,
The method according to (1C) above.
(11C) In step (E), the step of creating a group had {identical sequence determined based on sequence identity or similarity {preferably in the sequence of the molecular barcode portion} {eg, It is carried out by creating a group by clustering molecules presumed to have a sequence change by any of the steps (A) to (D) as a group
The method according to (1C) to (10C) above.
(12C) In step (E), clustering is
(I) In the sequence of the molecular barcode portion, by grouping nucleic acid molecule groups {ie, Distance = 0} having the same sequence as the unique molecular barcode sequence into the same cluster;
(Ii) in the sequence of the molecular barcode portion, by grouping the unique molecular barcode sequence and the nucleic acid molecule group {ie, Distance = 1} having a sequence having a mismatch up to one base into the same cluster;
(Iii) in the sequence of the molecular barcode portion, by grouping nucleic acid molecule groups {i.e., Distance = 2} having a sequence of unique molecular barcode and a sequence having up to 2 bases of mismatch into the same cluster; Or (iv) in the sequence of the molecular barcode portion, by grouping nucleic acid molecule groups {i.e., Distance = 3} having a sequence with a unique molecular barcode sequence and a mismatch of up to 3 bases into the same cluster ,
The method according to (11C) above.
(13C) In step (E), clustering is
In the sequence of the molecular barcode portion, a nucleic acid molecule group having a sequence sequenced as having an insertion or deletion (indel) of bases (for example, up to 1 base, up to 2 bases, or up to 3 bases) is classified into the same cluster To be done by
The method according to (11C) or (12C) above.
(14C) In step (E), clustering is
A group of nucleic acid molecules obtained by excluding sequences sequenced as having insertions or deletions (indel) of bases (for example, up to 1 base, up to 2 bases, or up to 3 bases) in the sequence of the molecular barcode portion To be done against
The method according to (11C) or (12C) above.
(15C) One or more (eg, one, two, three, four, five or six) insertions or deletions of said bases are arranged in all molecular barcode sequences linked to the nucleic acid molecule The method according to any one of claims 13 or 14, further comprising specifying the difference in position between each of the fixed bases of the one or more) and the position of each of the fixed bases in the sequence of the sequenced molecular barcode portion. {E.g., each fixed base may be designed to be any one base selected from the group consisting of A, T, G and C; or a combination of A and T, A and T G combination, A and C combination, T and G combination, T and C combination, G and C combination, A, T and G combination, A and T and C combination, A and G and It consists of the combination with C, and the combination with T and G and C It may be designed to be a base selected at random from bases contained in any one combination selected from}.
(16C) A target nucleic acid molecule (eg, a nucleic acid molecule of interest) to which a unique index (index sequence nucleic acid molecule) and a molecular barcode unique to each nucleic acid molecule (barcode sequence nucleic acid molecule) are added , The number of target nucleic acid molecules contained in a specific original sample from sequence information obtained by sequencing (ie, multiplex sequencing) using a mixture of multiple samples containing DNA, RNA or How to determine
(A) separately obtaining a plurality of samples comprising a nucleic acid molecule (eg, DNA or RNA) and {at least one of the samples comprising the nucleic acid molecule of interest},
(B) A target nucleic acid molecule in which an arbitrary molecular barcode is linked to each of the target nucleic acid molecules in each of a plurality of obtained samples before amplification of a nucleic acid molecule contained in the sample, and different molecular barcodes are linked to each other Obtaining
(C) Before mixing a plurality of samples, add a unique index to the target nucleic acid molecule for each sample containing a plurality of target nucleic acid molecules, and obtain a library of target nucleic acid molecules in which different indexes are linked for each sample derived Step and (The order of Step B and Step C may be first; and after Step B or C, the nucleic acid molecule can be amplified to obtain the amplification product of the target nucleic acid molecule),
(D) in a mixture containing the amplification products of the nucleic acid molecule obtained after (B) and (C) above (the sample is mixed after step (C) and after the sample is mixed (B) ) May be performed, or all samples may be mixed after performing step (B), and it is after step (B) that the amplification product of the nucleic acid molecule to which the molecular barcode is linked is obtained. The sample may be mixed before obtaining the amplification product, and after obtaining the amplification product, the sample containing the amplification product may be mixed), an index unique to each sample, and a unique or optional for each target nucleic acid molecule Sequencing the nucleic acid molecules to which the molecular barcodes have been added to identify the sequence of the index portion, the sequence of the molecular barcode portion, and the sequence of the nucleic acid molecule portion linked thereto for each nucleic acid molecule;
(E) selecting a nucleic acid molecule containing the sequence of the target nucleic acid molecule from the obtained sequence information;
(F) clustering the nucleic acid molecules selected in (e) above for each unique molecular barcode sequence, and then identifying a cluster having a plurality of sequences in the index nucleic acid molecule portion,
(G) In each of the clusters identified in (f), the index with the highest detection frequency and the molecular barcode pair are identified as the correctly indexed target nucleic acid molecules, and the other index and molecular barcode pairs are identified. Determining that it is a mispair, and
{Wherein it may further include determining that the index is incorrect in Mispair},
The target nucleic acid molecule contained in the sample corresponding to the index, the number of kinds of sequence of the unique molecular barcode linked to the target nucleic acid molecule correctly indexed (or the number of clusters of the target nucleic acid molecule correctly indexed) Is the number of
Method.
(17C) In (f), clustering is
(I) In the sequence of the molecular barcode portion, by grouping nucleic acid molecule groups having the same sequence as the unique molecular barcode sequence into the same cluster;
(Ii) in the sequence of the molecular barcode portion, by grouping nucleic acid molecule groups having a unique molecular barcode sequence and a sequence having up to one mismatch into the same cluster;
(Iii) in the sequence of the molecular barcode portion, by grouping nucleic acid molecules having a unique molecular barcode sequence and a sequence having up to 2 bases of mismatch into the same cluster; or (iv) the molecular barcode In the partial sequence, it is performed by classifying nucleic acid molecule groups having a sequence having a unique molecular barcode and a mismatch up to 3 bases into the same cluster,
The method as described in the above (16C).
(18C) In (e), clustering is
In the sequence of the molecular barcode portion, a group of nucleic acid molecules having a sequence sequenced as having an insertion or deletion (indel) of bases (for example, up to 1 base, up to 2 bases, or up to 3 bases) in the same cluster Done by classification,
The method according to (16C) or (17C) above.
(19C) In (e), clustering is
A nucleic acid molecule obtained by excluding sequences sequenced as having insertions or deletions (indel) of bases (eg, up to 1 base, up to 2 bases, or up to 3 bases) in the sequence of the molecular barcode portion Performed on a group,
The method according to (16C) or (17C) above.
(20C) One or more (eg, one, two, three, four, five or six) insertions or deletions of said bases are arranged in all molecular barcode sequences linked to the nucleic acid molecule 20. The method according to claim 18 or 19, further comprising: identifying by the difference between the positions of each of the fixed bases (or more) and the position of each of the fixed bases in the sequence of the sequenced molecular barcode portion. {E.g., each fixed base can be designed to be any one base selected from the group consisting of A, T, G and C; or a combination of A and T, of A and G Combination, A and C combination, T and G combination, T and C combination, G and C combination, A, T and G combination, A, T and C combination, A, G and C and And the combination of T, G and C from It may be designed to be a base selected at random from bases contained in any one of the combinations to be-option}.

FIG. 1 is a diagram for explaining a digital quantification method of nucleic acid molecule and its effectiveness. (A) In panel A of FIG. 1, a scheme of digital counting is shown. Uniquely add a molecular barcode to each target nucleic acid molecule (add a unique molecular barcode). After amplification, both the nucleic acid portion of interest and the barcode portion are sequenced. The copy number is determined not by the read number but by the number of unique barcodes. The dotted frame indicates the experimental design used in this example. (B) Panel B of FIG. 1 illustrates that the first requirement for accurate digital counting: each target nucleic acid molecule must be labeled with a different barcode. As the number of unique barcodes measured becomes constant as the diversity of barcode sequences increases, the diversity of barcode sequences in the range meets the first requirement. (C) Panel C of FIG. 1 illustrates that the second requirement for accurate digital counting: all barcode sequences (at least one lead) attached to the target nucleic acid molecule must be detected FIG. If the number of unique barcodes measured becomes constant as the sequence depth is increased, then the sequence depth of the range meets the second requirement. FIG. 2 shows the observed intrinsic features of digital counts with random base barcodes applied to the two requirements for accurate digital quantification. (A) Panel A of FIG. 2 represents the dependence of the number of clusters (unique barcodes shown in gray) on the number of random bases (base length). The results of ST1 are shown. Gray lines indicate the number of unique barcodes. The blue line shows the number of observed clusters after clustering (Distance = 3). The green line shows the number of clusters after clustering (Distance = 3) and fixed base match filtering (number of fixed bases = 6). (B) Panel B of FIG. 2 shows the dependence of the number of barcode clusters on the number of random bases. The yellow line indicates the number of clusters for ST1 with index A and index B after clustering (Distance = 3) and fixed base match filtering (number of fixed bases = 6). Red dotted lines indicate the number of barcode clusters for all short templates with index A and index B after clustering (Distance = 3) and fixed base match filtering (fixed base number = 6). (C) Panel C in FIG. 2 shows the dependence of the number of detected molecules on the sequence coverage. Gray lines represent the dependence of the number of unique barcode sequences observed on the number of random bases. The blue line shows the number of observed clusters after clustering (Distance = 3). The green line shows the number of clusters after clustering (Distance = 3) and fixed base match filtering (number of fixed bases = 6). The yellow line indicates the number of barcode clusters for ST1 with index A after clustering (Distance = 3), fixed base match filtering (fixed base number = 6), and exclusion of miss index (eg, contamination index) Show. Red dotted line indicates clustering (STD = 3), fixed base match filtering (fixed base number = 6), and bar code cluster for ST1 with index A after miss index (eg contamination index) and misidentification exclusion Indicates the number. Error bars indicate standard deviations (n = 8). FIG. 3 shows the results of analysis using Distance and fixed base. In FIG. 3, the results of ST1 with index A (indicated by circles) and index B (indicated by triangles) are shown. The length of the random base barcode was 24. (A) Panel A of FIG. 3 shows the effect of clustering at different Distances on the number of clusters. (B) Panel B in FIG. 3 shows the dependence of the position of the fixed base at Distance = 3. Asterisks indicate no filtering. Filtering was done using only one fixed base. (C) Panel C in FIG. 3 shows the dependence of the number of fixed bases at Distance = 3. Asterisks indicate no filtering. The filtering was done using the base farthest from the sequence primer site. FIG. 4 shows the absolute counts of each template. (A) Panel A of FIG. 4 shows the number of clusters determined for index A as a function of coverage. Distance = 2, number of fixed bases = 4, number of random bases = 20. In addition, the effects of miss index (eg, contamination index) and the effects of misidentification were excluded. The starting copy number of each template sequence is indicated in parentheses. (B) Panel B of FIG. 4 shows the dependence of the number of detected molecules on the number of random bases. Distance = 2, number of fixed bases = 4. Ten percent of the total reads were randomly sampled (coverage for short molds was 13.4 to 20.3 and coverage for long molds was 12.6 to 20.9). (C) Panel C in FIG. 4 shows the correlation between the input (ie, the number of molecules before PCR amplification, x axis) and the output (ie, the result of digital counting, the y axis). The number of outputs was determined from FIGS. 4A and 11 with a coverage of 12.6 to 20.9 indicated by the large symbols. The gray line shows a regression line with slope 1 on a logarithmic scale. The circles and triangles correspond to index A and index B, respectively. Pearson's product moment correlation coefficient r of linear regression and determination coefficient R ² are shown. Error bars indicate standard deviations (n = 8). FIG. 5 shows the required number of random bases for digital counting. (A) In panel A of FIG. 5, the x-axis indicates the number of inputs of the molecule to be measured, and the y-axis is a relative of 0.95 for each curve in panel B of FIG. 4 and panel B of FIG. Indicates the number of random bases when reaching the target number of clusters. (B) Panel B of FIG. 5 shows the dependence of the number of barcode clusters on the number of random bases. Clustering (Distance = 2), all templates with indexes A and B after fixed base match filtering (fixed base number = 4), randomly sampling 10% of all reads (for example, the coverage is 12.6-20.9). The colors correspond to the samples represented by the plots in panel A of FIG. Error bars represent standard deviations (n = 8). FIG. 6A shows a conventional method involving amplification, indexing, mixing and sequencing. In FIG. 6A, the barcoded sequence is not used, and a unique index is added to the amplified sequence for each sample, mixed and sequenced. An index may be added before amplification. FIG. 6B shows a conventional method involving amplification, indexing, mixing and sequencing. Here, in the conventional method, although miss index addition may occur, the generated miss index can not be identified. FIG. 6C shows the use of molecular barcodes. A unique barcode sequence is labeled for each nucleic acid sequence of interest having sequence 1, and each molecule is uniquely labeled. FIG. 6D illustrates the use of molecular barcodes, including molecular barcode addition, amplification, indexing, mixing and sequencing. Unique barcode attachment to nucleic acid molecules and unique index is added to each sample, showing a scheme for mixing and sequencing multiple samples. An index may be added after amplification of the molecular barcodes, but before amplification. FIG. 6E illustrates the use of molecular barcodes, including molecular barcode addition, amplification, indexing, mixing and sequencing. 1 shows a scheme of an example of a method of identifying a miss index in the first embodiment of the present invention. Although a miss index may occur, in the first embodiment of the present invention, the encountered miss index can be identified. It is assumed that amplification products from the same molecule have the same index and the number of misindexed molecules is smaller than the number of correctly indexed molecules. In the first embodiment of the present invention, when two types of indexes are added to the same barcode arrangement, the most frequent index in the number of reads is determined as the correct index. FIG. 7A shows a scheme for adding a barcode to a target nucleic acid molecule contained in a plurality of samples. FIG. 7B shows a scheme of indexing and amplification, and shows the case in which partial switching occurs due to indexes that are contaminated with other indexes. FIG. 7C shows the count of the bar code number, confirmation of the same bar code, and identification of an error (index and bar code mispair). The scheme which identifies the molecule | numerator with few read numbers (copy number) as a miss index is shown, when different index A and B are added with respect to the same barcode arrangement | sequence. FIG. 8 is a supplementary drawing of FIG. 2 and shows the observed intrinsic features of digital counts using barcodes with random bases. (A) Panel A of FIG. 8 represents the dependence of the number of clusters detected on the number of random bases (base length) for ST1, ST2, LT1 and LT2. Gray lines represent the dependence of the number of unique barcode sequences observed on the number of random bases. The blue line shows the number of observed clusters after clustering (Distance = 3). The green line shows the number of clusters after clustering (Distance = 3) and fixed base match filtering (number of fixed bases = 6). (B) Panel B of FIG. 8 shows the dependence of the number of barcode clusters on the number of random bases. Yellow lines indicate clustering (Distance = 3), fixed base match filtering (number of fixed bases = 6 for ST2, number of fixed bases = 12 for LT2), miss index (eg, contamination error index) The number of clusters for ST2 (upper panel) and LT2 (lower panel) with indices A and B after exclusion is shown. Dark yellow lines indicate clustering (distance = 3), fixed base match filtering (fixed number of bases = 12), LT1 with index A and index B after exclusion of miss index (eg contamination index) (lower panel) Indicates the number of clusters for. Red dotted lines indicate all long with index A and index B after exclusion of clustering (distance = 3), fixed base match filtering (fixed number of bases = 6), miss index (eg contamination index) and misidentification Indicates the number of barcode clusters for the template. (C) Panel C of FIG. 8 shows the dependence of the number of molecules detected on the sequence coverage for ST1, ST2, LT1 and LT2. In FIG. 8 panel C, the color of the line is the same as panels A and B in FIG. Error bars indicate standard deviations (n = 8). FIG. 9 is a supplementary drawing of FIG. 3 and shows the analysis results using Distance and fixed base for ST2, LT1 and LT2. (A) Panel A of FIG. 9 shows the effect of clustering on the number of clusters as in panel A of FIG. 3 but for ST2, LT1 and LT2 with different Distance parameters. (B) Panel B of FIG. 9 is the same as panel B of FIG. 3, but shows the dependence of the position of the fixed base on ST2, LT1 and LT2. Asterisks indicate no filtering. (C) Panel C of FIG. 9 is the same as panel C of FIG. 3, but shows the dependence of the number of fixed bases on ST2, LT1 and LT2. FIG. 10 shows a histogram of the number of reads in each cluster for ST1 with index A (see panel A) and index B (see panel B). The colors correspond to the sample colors of the plot of FIG. 2C. FIG. 11 is a supplementary drawing of FIG. 4 and shows the absolute count of each mold. The same as in panel A of FIG. 4, but for index B (indicated by triangles) indicates the number of clusters determined for coverage. Error bars indicate standard deviations (n = 8). FIG. 12 is a supplementary drawing of FIG. 5 and shows an estimation of the required number of random bases for digital counting. This plot is the same as FIG. 5A except that it is a linear regression (R ² = 0.971) on a logarithmic scale up to random base number 38 (longest in this example). The equation for the regression curve above in this case was y = 2.2 * log (x) +5.5. FIG. 13 shows the bar code design and the number of numerator inputs. The capital letters in the sequences of LT1 to 6 are binding sites of PCR amplification primers. Barcode indicates a random region including random bases and fixed bases, and target indicates a target nucleic acid. LT1-6 were PAGE purified products, and the 5 'end of ST1-5 was amine-modified. The fixed base between the random bases helps to avoid long homopolymeric barcodes which may have lower amplification efficiency. N represents any one of A, T, G, or C. FIG. 14 shows primer sequences for preparation of the library. The underlined part indicates an index array (index A is included in Rv primer 1 and index B is included in Rv primer 2). All primers were PAGE purified. FIG. 15 shows the number of leads in each process. * This fraction may be greater than the number of leads in the exclusion of a miss index (eg, contamination index) (see example).

Detailed Description of the Invention

As used herein, a "molecular barcode" is a tag having a unique sequence that is attached to each molecule of nucleic acid. Also called "primer ID" and "Ubiquitous Molecular Identifier (UMI)". When a molecular barcode having a unique sequence that differs from molecule to molecule is added to a nucleic acid molecule, the number of molecules of the nucleic acid contained in the sample before being subjected to processing such as amplification is added. It can be determined digitally (or qualitatively) based on the number of bar code types. This method of determination of nucleic acid molecules has attracted a great deal of attention as a next-generation sequencer platform that has made it possible to analyze a large amount of nucleic acid sequences in a single run, and has become a focus of attention. Various methods have been developed to determine the number digitally. Since this method of determining the number of nucleic acid molecules can digitally count the number of molecules as the number of types of barcodes (sometimes referred to as the “number of unique barcodes”), “digital counting method” or “digital quantification method” It is sometimes called "etc." This digital counting method can accurately determine the absolute number of molecules in a sample, even in the presence of the noise and bias of the measurement system. The most widely used application of this digital counting method is RNA-Seq using molecular barcodes, ie, digital RNA-Seq (dRNA-Seq) or quantitative RNA-Seq. dRNA-Seq is often used for single cell gene expression analysis because it works well even with a small amount of sample.
Digital counting is also used for many applications in next generation sequencer platforms that can acquire large amounts of sequence data. As such applications, for example, in addition to RNA-Seq, single nucleotide resolution UV cross-linked immunoprecipitation (iCLIP: individual cross-link resolution and UV cross-linking and immunoprecipitation), antibody repertoire analysis, genetic analysis of bacterial 16S rRNA, And exonucleases, unique barcodes and single ligation-mediated nucleotide resolution chromatin immunoprecipitation experiments (ChIP-nexus: chromatin immunoprecipitation experiments with nucleotide resolution through exonuclease, unique barcode and single ligation).
In this method of digital counting, by using a sufficiently large number of types of molecular barcodes relative to the total number of nucleic acid molecules present in the sample, the same barcode can be applied to multiple nucleic acid molecules present in the original sample. The possibility of addition is substantially limited, which allows the number of types of barcode sequences to correspond to the number of nucleic acid molecules present in the sample. In this way, quantification of nucleic acid molecules present in the sample is possible by using molecular barcodes comprising nucleotide sequences with sufficient diversity. Molecular barcodes can be obtained, for example, as nucleic acid groups with random bases. Since the molecular barcode is focused on the number of types of its sequence to determine the number of molecules to be measured, the sequence is random (the sequence is diverse and it is not necessary for humans to recognize the content of the sequence It may be said that they may be synthesized in the same manner. Alternatively, the molecular barcode may be a set of nucleic acids of known sequence designed to obtain sufficient diversity. In the present specification, a molecular barcode may be simply referred to as a barcode, and a sequence possessed by the molecular barcode may be referred to as a barcode sequence. As used herein, the number of unique barcode sequences is a number that represents the degree of diversity of barcode sequences. The number of unique barcode sequences is n if n different barcode sequences are detected {where n is a natural number}. As used herein, the number of random bases means the base length of random bases. As used herein, a random base refers to a continuous base having a random sequence. The random bases may consist of two bases, three bases or four bases.

As used herein, an "index" is a nucleic acid that serves as a unique label attached to a nucleic acid molecule for each sample from which it is derived. For example, an index can be added that has a different nucleotide sequence for each sample. When a plurality of samples are mixed and sequenced by adding the same index to all nucleic acid molecules derived from a certain sample, samples from which individual nucleic acid molecules are derived based on the sequence of the added index It can be identified. Due to the large capacity of one sequencing on the platform of the next-generation sequencer, it is possible to mix multiple samples and sequence them in one run, for example, the index is useful in such a case is there. The addition of the index may be added before, during or after processing (eg, amplification) of the nucleic acid molecule.

As used herein, the terms "template", "target nucleic acid", "target nucleic acid molecule", "target nucleic acid" or "target nucleic acid molecule" refer to a nucleic acid molecule (eg, DNA or RNA) to be quantified in digital quantification. Can be used interchangeably. In the present specification, the sequence originally possessed by the target nucleic acid molecule (ie, the sequence before the barcode or index is added for analysis) is referred to as the target nucleic acid sequence.

As used herein, "nucleic acid" means a macromolecule having a nucleic acid sequence. Nucleic acids include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The ribonucleic acid includes messenger RNA (mRNA), non-coding RNA such as microRNA, transfer RNA (rRNA), and ribosomal RNA (rRNA).

As used herein, "sequence depth" refers to the total amount or total number of molecules to be sequenced. For example, high sequence depth (ie, more sequence information can be obtained) may increase the likelihood that sequences only slightly present in the sample will be detected. As used herein, “coverage” means the average number of reads of each cluster (read number / cluster) obtained by clustering as originating from the same nucleic acid molecule.

As used herein, "specific to each molecule" means that they differ from one another for at least a portion of the molecules contained in the system. The “specific to each molecule” means all molecules contained in the system, substantially all molecules, or most of the molecules (eg, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more) % Or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more).

The conventional procedure of digital quantification of nucleic acids is described below (see panel A of FIG. 1).
Uniquely adds DNA (molecular barcodes) containing various foreign sequences to each of nucleic acids (target nucleic acid molecules) such as RNA molecules or DNA (eg, complementary DNA or cDNA) molecules (ie, nucleic acids Add molecular barcodes with different sequences for each molecule) (see, eg, FIG. 6C). Such a nucleic acid to which a molecular barcode having a unique sequence for each molecule is added may be referred to as a "barcoded nucleic acid". The barcoded target nucleic acid molecule (cDNA obtained from RNA if the starting nucleic acid is RNA) is amplified (see, eg, FIG. 6D). The target nucleic acid sequence of the barcoded and amplified nucleic acid and the barcoded sequence are sequenced in tandem (see, eg, FIG. 6D). As theoretically proposed, for each target nucleic acid, the number of unique barcodes attached to the target nucleic acid sequence is quantified rather than the number of amplified molecules (the so-called "read number"), and the original The absolute copy number of the nucleic acid molecule of interest (before amplification) can be determined. In this digital quantification method, since the number of types of barcode sequences is focused, the barcode sequence may be added to the target nucleic acid molecule so as to have a unique sequence for each nucleic acid molecule, and the specific sequence thereof It does not matter what the In digital quantification, barcodes whose specific sequence is known may be used.
The platform of the next generation sequencer has been developed, and it has become possible to decode a large number of base sequences by one sequencing (run). As a result, single-sample measurements do not run out of sequencing capabilities and the need for sequencing multiple samples simultaneously in a single run is growing. While sequencing multiple samples in a single run, a unique index can be added to each sample to distinguish which sample the nucleic acid came from. According to the present invention, the index may be added to the target nucleic acid molecule so as to have any sequence unique to each sample, regardless of the specific sequence. In digital quantification, an index whose specific sequence is known may be used.
According to the present invention, the index may be added to the amplified target nucleic acid molecule after the target nucleic acid molecule is amplified, or added to the target nucleic acid molecule before the target nucleic acid molecule is amplified. It may be done. An index may be added after performing amplification on each sample. For example, the addition of an index can be performed on each of the amplification products by adapter ligation. Alternatively, the index may be added while the nucleic acid molecule of interest is amplified. For example, the addition of an index can be performed during amplification of the nucleic acid molecule by including it in the sequence of the primer.
In the present invention, when the index is added to the target nucleic acid molecule before amplification, the index may be added to the target nucleic acid molecule before, simultaneously with or after the addition of the barcode sequence. The index, the barcode sequence, and the nucleic acid molecule of interest may be linked in any order. The index may be provided in conjunction with the barcode array. When subjecting a target nucleic acid molecule contained in a specific sample to digital quantification using molecular barcodes, the target nucleic acid molecule derived from the specific sample can be identified using the index as an index, and is added to the target nucleic acid sequence The number of unique barcode sequences (number of unique barcodes) is quantified to determine the absolute copy number of the original (before amplification) nucleic acid molecule of interest (see, eg, FIG. 6D).

According to the present invention, in the method for digitally quantifying a target nucleic acid molecule using an index and a barcode, when a plurality of samples are mixed and the target nucleic acid molecule is quantified, a nucleic acid derived from a sample having an unexpected index It has become apparent that problems may arise that are added to (see FIG. 6E, FIG. 7B). This problem may occur when using an index, and is referred to as index switching, index hopping, misindexing, and the like. The existence of the index switching problem has already been pointed out (Sinha, R. et al. Index switching causes “spreading-of-signal” among multiplexed samples in Illumina HiSeq 4000 DNA sequencing. Biorxiv, 10.1101 / 125724 (2017)) To date no effective solution has been reported.
According to the invention, also, when counting the number of types of barcode sequences, sequences that differ as to be judged to be the same due to mutations (eg, insertions, substitutions, and deletions) that occur within the barcode sequence It has become apparent that problems can be encountered as perceived. These problems can occur with or without the use of indexes.

The present invention provides solutions to each of these problems.
In a digital quantification method that uses a sample-specific index to distinguish samples, multiple bar codes and indexes are not added to the same bar code for the target nucleic acid molecule to which the index is added. (Since each nucleic acid molecule has a unique barcode attached). On the other hand, in the present invention, when multiple indexes are found in a cluster of nucleic acid molecules to which the same barcode is added, it can be determined that a miss index has occurred (for example, FIG. 6E). And Figure 7C). When multiple indexes are found in a cluster of nucleic acid molecules to which the same barcode is added, the number of existing index sequences is compared, and the most frequently occurring index sequence is the correctly indexed sequence. It is determined that there is (see, for example, FIGS. 6E and 7C). This can correspond to a miss index (eg, by excluding sequences other than the most frequent index sequence in one cluster). It goes without saying that this method can be carried out independently of the sequence of the target nucleic acid molecule. Thus, the method may or may not include decoding the nucleic acid sequence of interest. This method corresponds to the first embodiment described below.

In the digital quantification method, the index sequence and the barcode sequence have different identifications that affect the accuracy of quantification. For example, if the barcoded sequence is recognized as a different sequence by mutation (for example, insertion, substitution, and deletion) of bases in the sequence regardless of whether or not an index is added, the number of sequence types is The digital quantification used to determine the original number of molecules before being subjected to amplification etc. makes the determination of the number of molecules inaccurate. On the other hand, in the present invention, for substitution of bases in a barcode, the sequences included in a certain distance (Distance) are clustered as one cluster, and the number of molecules is determined based on the number of clusters. The substitution of bases can address the problem of being recognized as an identical but different sequence. Here, "distance" refers to the number of bases that differ between two predetermined barcode sequences. For example, if one barcode sequence is exactly the same as another barcode sequence except for one base change at any one position, the distance between these two barcode sequences is 1 is there. For example, the distance between these two barcode sequences is 2 if exactly the same except for the two base changes at any two positions. For example, if one barcode sequence is exactly the same as another barcode sequence except for three base changes at any three positions, then the distance between these two barcode sequences is 3 It is. It is believed that the accuracy of the method of the first embodiment increases as the diversity of barcode sequences increases. The value of Distance is not particularly limited as long as it is appropriately determined according to the present disclosure, but is, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3, and still more preferably 3. It goes without saying that this method can be carried out independently of the sequence of the target nucleic acid molecule. Thus, the method may or may not include decoding the nucleic acid sequence of interest. This method corresponds to the second embodiment described below. In a system for adding an index, it can be used similarly to determine the difference between the indexes.

Also, for example, for insertions and deletions of bases in a barcode sequence (for insertions and deletions sometimes collectively referred to as "indel"), the barcode may or may not be added with an index. By setting the base at the fixed position as the fixed base (ie, setting the base at the predetermined position in the barcode sequence as the specific or defined base), generation of indel is indicated by the absence of the fixed base at the predetermined position. (This method may be referred to herein as "fixed base match filtering"). That is, it is determined that insertion or deletion of a base has occurred in the barcode sequence when the sequenced barcode sequence contains a base different from the original base at any of the fixed base positions. The number of fixed bases in the barcode sequence is not particularly limited as long as it is appropriately determined according to the present disclosure, but it is, for example, 1 to 15, preferably 2 to 12, more preferably 3 to 10, still more preferably 4 to There are six. It goes without saying that this method can be carried out independently of the sequence of the target nucleic acid molecule. Thus, the method may or may not include decoding the nucleic acid sequence of interest. This method corresponds to the third embodiment described below. In a system for adding an index, it can be used similarly to determine the difference between the indexes.

Hereinafter, each of the first embodiment, the second embodiment, and the third embodiment will be described. Note that these embodiments can also be implemented in combination, and the present invention includes combinations of such possible embodiments. The following embodiments are intended to include non-limiting examples of combined embodiments.

A first embodiment of the present invention That is, according to the first embodiment of the present invention,
Sequencing using a mixture of multiple samples containing the nucleic acid molecule of interest with an index unique to each sample containing multiple nucleic acid molecules and a unique or arbitrary molecular barcode unique to each nucleic acid molecule (multiplex sequencing) A method of determining the correct pair or mispair of an index added to a target nucleic acid molecule and a molecular barcode from sequence information obtained by
(E) From the obtained sequence information, a sequence having a specific index or a sequence similar thereto, a sequence having a specific molecular barcode or a sequence similar thereto, or a sequence containing a target nucleic acid molecule or a similar Selecting a sequence and creating a group from the selected sequence;
(F) In the group created in (E) above, determine the pair with the highest detection frequency index and molecular barcode as the correct pair of index and molecular barcode, and / or the low detection frequency index and molecules Bar code pair (for example, a detection frequency pair lower than a predetermined reference value, and the predetermined reference value is 99.5% or less, 99% or less, 90% or less, 80% or less, 70% or less, 60% or less) % Or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 1% or less, but is not limited thereto. Determining at least any one or all of the frequency pairs) as the index and the molecule barcode mispairing;
Methods are provided.

In a first embodiment of the invention, the method of the invention
(A) separately obtaining a plurality of samples containing a nucleic acid molecule (eg, DNA or RNA) and {at least one of the samples includes a target nucleic acid molecule},
(B) {e.g., in each of a plurality of obtained samples} before amplification of the nucleic acid molecules contained in the sample, each nucleic acid molecule of interest is linked to each of the nucleic acid molecules, or each molecular barcode unique to each nucleic acid molecule is linked; Obtaining a target nucleic acid molecule to which different molecular barcodes are linked;
(C) {For example, before mixing a plurality of samples} A target nucleic acid molecule in which a unique index is added to the target nucleic acid molecule for each sample containing a plurality of target nucleic acid molecules, and different indexes are linked for each sample derived And (step (C) may be performed after step (B), or step (B) may be performed after step (C); and step (B) or (C)). After the nucleic acid molecule can be amplified to obtain an amplification product of the target nucleic acid molecule),
(D) in a mixture containing the amplification products of the nucleic acid molecule obtained after (B) and (C) above (the sample is mixed after step (C) and after the sample is mixed (B) ) May be performed, or all samples may be mixed after step (B), and it is after step (B) that the amplification product of the nucleic acid molecule to which the molecular barcode is linked is obtained. The sample may be mixed before obtaining the amplification product, and after obtaining the amplification product, the sample containing the amplification product may be mixed), an index unique to each sample, and a unique or optional for each target nucleic acid molecule And sequencing the nucleic acid molecule to which the molecular barcode of S is added to determine the sequence of the index portion, the sequence of the molecular barcode portion, and the sequence of the target nucleic acid molecule portion linked thereto as needed for each nucleic acid molecule. When,
It may further include a.

In the first embodiment of the present invention, any index may be used as long as it has a unique base sequence for each sample. The index may have a predetermined sequence (eg, it may be possible to determine which sample is derived from by referring to the sequence), but the sequence is unknown. (For example, reference to a sequence makes it impossible to determine which sample it is derived from, and it may be known that different sequences lead to different samples).

In a first embodiment of the invention, molecular barcodes can be made to have sufficient diversity to the number of nucleic acid molecules in the sample. The molecular barcode may have any base sequence as long as it has sufficient diversity to the number of nucleic acid molecules in the sample. The sequence of the molecular barcode can be a randomly determined sequence (a randomly determined sequence), for the purpose of saving time and effort of sequence design. For example, the molecular barcode may have sufficient diversity by including a plurality of randomly determined bases (ie, random bases). In order to ensure the diversity of molecular barcodes, the length of the base sequence of molecular barcodes can be increased. When random bases are used in digital quantification of a target nucleic acid having a predetermined diversity, the number of random bases in the base sequence of the required molecular barcode is determined experimentally based on the graph as exemplified in FIG. You may Although the present invention is not limited, for example, by setting the number of random bases in the base sequence of the molecular barcode to 38 or more, sufficient diversity is secured for digitally quantifying the number of molecules covering 10 ¹⁵ It can be understood from the examples that can be done. If the four bases are randomly arranged and the base length is 38, then the diversity of the molecular barcodes theoretically extends to 4 ³⁸ (ie, about 7.56 × 10 ²² ). The number of random bases in the molecular barcode is, for example, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 12 or more, 13 or more, 14 or more, 15 or more to ensure sequence diversity. , 16 or more, 17 or more, 18 or more, 19 or more, or 20 or more. Alternatively, the number of random bases may be 25 or more, 30 or more, 35 or more, 40 or more.

In the first embodiment of the present invention, the plurality of samples are 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more samples, and It is a distinguishable number, but there is no upper limit to the number.

In the first embodiment of the present invention, in the above (E), a sequence having a specific index, a sequence having a specific molecular barcode, and / or a sequence containing a target nucleic acid molecule based on sequence identity Groups can be formed by the sequences selected and selected. Here, by selecting a sequence having a specific molecule barcode and forming a group for each molecular barcode by the selected sequence, it is possible to form a number of groups corresponding to the number of types of molecular barcodes. . Also, by selecting an array having a specific index and forming a group for each index according to the selected array, a number corresponding to the number of indexes (for example, the number of samples when adding a different index for each sample) Can form a group of Also, by selecting a sequence having a specific target nucleic acid and forming a group with the selected sequence, a nucleic acid group including the target nucleic acid can be obtained.

In the first embodiment of the present invention, the step of (E) above comprises creating identical groups {preferably in the sequence of the molecular barcode portion} based on sequence identity or similarity This can be performed by creating a group by clustering molecules presumed to be {eg, the sequence may be changed by any of the steps (A) to (D)} as a group.
In the first embodiment of the present invention, for example, the above (E) can be implemented in combination with the second embodiment. Details will be described in the second embodiment.
Furthermore, in the first embodiment of the present invention, for example, the above (E) can be implemented in combination with the second embodiment and the third embodiment. Details will be described in the third embodiment.

In the first embodiment of the present invention, in the above (F), for each of the groups created in the above (E), the index with the highest detection frequency and the pair of molecular barcodes are the index and the correct pair of molecular barcodes and It can be decided. In the first embodiment of the present invention, at least any one or all of the low detection frequency index and molecular barcode pairs may be determined as the index and molecular barcode mispairing. In the first embodiment of the present invention, the highest detection frequency index and molecular barcode pair is determined as the correct index and molecular barcode pair, and at least low frequency detection index and molecular barcode pair Any one or all may be determined as the index and the misplacement of the molecular barcode. In a first embodiment of the invention, nucleic acid molecules determined to be mispaired can be excluded from the count of the number of molecules. Determination of the correct pair and determination of the mispair, respectively, can be performed regardless of the sequence of the nucleic acid molecule of interest. For example, the target nucleic acid molecule may be selected to determine the correct pair and the mispair, respectively; or the target nucleic acid molecule may be selected after determining the correct pair and the mispair. You may

For example, in one embodiment, in the above (E), when a sequence having a specific molecular barcode is selected to create a group for each molecular barcode,
(i) In step (F), the pair with the highest detection frequency index and molecular barcode among the created groups can be determined as the correct pair of index and molecular barcode; or
(ii) Among the groups created in step (F), a pair with a low detection frequency index and a molecular barcode (for example, a pair with a detection frequency lower than a certain reference value Values include 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, and 1% or less, and are not limited to these. May be determined as an index and / or a molecule barcode and / or a mispair of at least one or all of them.

For example, in one embodiment, in (E) above, when an array having a specific index is selected and a group is created for each index,
(iii) In the step (F), for nucleic acid sequences having molecular barcodes appearing in a plurality of groups, determine the barcode / index pair with the highest number of reads as the correct barcode / index pair, or The highest detection frequency index and molecular barcode pair can be determined as the correct index and molecular barcode pair; or
(iv) In the step (F), a low detection frequency index and molecular barcode pair (for example, a detection frequency pair lower than a predetermined reference value) for sequences having molecular barcodes appearing in a plurality of groups The certain reference value includes, but is not limited to, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 1% or less in the group. The second and subsequent detection frequency pairs may be determined as) at least any one or all of the indices and the mispair of the molecular barcode.

For example, in one embodiment, in (E) above, when a sequence including the target nucleic acid molecule is selected to create a group,
(v) In step (F), a sequence having a specific index is further selected from the group to create a subgroup, and the nucleic acid sequence having a molecular barcode appearing in a plurality of subgroups has the highest number of reads The barcode and index pair can be determined as the correct barcode and index pair, or the most frequently detected index and molecular barcode pair can be determined as the correct index and molecular barcode pair;
(vi) In the step (F), further select a molecule having a specific molecular barcode from the group to create a subgroup, and in the created subgroup, the index with the highest detection frequency and the molecular barcode The pair can be determined as the correct pair of index and molecular barcodes;
(vii) In the step (F), molecules having a specific index are further selected from the group to create subgroups, and nucleic acid molecules having molecular barcodes appearing in a plurality of subgroups are detected with an index with low detection frequency and Molecular barcode pairs (for example, a detection frequency pair lower than a certain reference value, which means 50% or less, 40% or less, 30% or less, 20% or less, 10% or less in the group) 5% or less, 1% or less, but not limited thereto, and may be, for example, a pair of second or later detection frequency) and at least any one or all of index and molecular barcode Can be determined as
(viii) In step (F), molecules having a specific molecular barcode are selected from the group to create subgroups, and a pair of low detection frequency index and molecular barcodes in one created subgroup ( For example, a pair with a detection frequency lower than a certain reference value, and the certain reference value means 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 1% The following values may be included, but are not limited thereto, and, for example, at least any one or all of the following may be detected as the index and the molecular barcode mispair: Can.

In this way, in the first embodiment of the present invention, the correct pair of barcode and index sequences can be determined and / or mispairing can be determined. As shown in Examples described later, not counting mispairs can improve the accuracy of digital quantification of a target nucleic acid molecule.

Second embodiment of the present invention In digital quantification of nucleic acid molecules using barcode sequences, mutations (insertions, substitutions or deletions) occur in the barcode sequences during analysis, and the mutations have a quantitative accuracy. It became clear that the A second embodiment of the present invention is a method for digitally quantifying a target nucleic acid molecule using a barcode sequence, and based on the obtained information on the nucleic acid sequence, has sequence similarity to the sequence possessed by the molecular barcode after mutation. It is related to grouping into 1 group together with the sequence of (clustering). This seeks to minimize the effects of mutations in barcode sequences that occur during analysis. In the second embodiment, for example, in an environment where it is less likely to contain sequences similar to molecular barcodes, similar sequences may be generated by mutation (insertion, substitution or deletion) from the same sequence. It is suggested that this clustering actually improves the accuracy of digital quantification even in the example.
More specifically, for example, the step of creating a group had the same sequence {determined preferably in the sequence of the molecular barcode portion} sequence identity or similarity {e.g. When (A) to (D) are performed, it may be possible to create a group by clustering molecules presumed to have a sequence mutation at any of these steps as a group. Therefore, a sequence having similarity with a sequence having a particular index includes a sequence having a particular index and a sequence having similarity to a sequence having a particular index.

In the second embodiment of the present invention, for example, in digital quantification of a target nucleic acid molecule using a barcode sequence, the obtained nucleic acid sequence is indexed, based on sequence similarity, a barcode and / or a target nucleic acid molecule Can be grouped (clustered). In an aspect of the second embodiment of the present invention, for example, clustering is:
(I) In the sequence of the molecular barcode portion, nucleic acid molecules having the same sequence as the unique molecular barcode sequence are classified into the same cluster (ie, Distance = 0);
(Ii) in the sequence of the molecular barcode portion, by grouping nucleic acid molecule groups having a sequence having a unique molecular barcode sequence and a mismatch of up to 1 base into the same cluster (ie, Distance = 1);
(Iii) In the sequence of the molecular barcode portion, by grouping nucleic acid molecule groups having a sequence of unique molecular barcode and a sequence having up to 2 bases of mismatch into the same cluster (ie, Distance = 2); Or (iv) in the sequence of the molecular barcode portion, by grouping nucleic acid molecule groups having a sequence with a unique molecular barcode sequence and a mismatch up to 3 bases into the same cluster (ie, Distance = 3) . In this way, an artificial increase in the type of nucleic acid sequence due to a 0-3 base mutation that can occur in digital quantification is corrected.
When this aspect of the second embodiment is combined with the first embodiment, it can be implemented in the step (E) above.

In the second embodiment of the present invention, for example, in digital quantification of a target nucleic acid molecule using a barcode sequence, the obtained nucleic acid sequence is indexed, based on sequence similarity, a barcode and / or a target nucleic acid molecule Can be grouped (clustered). In an aspect of the second embodiment of the invention, the clustering may for example be an insertion or deletion (indel up to 1 base, up to 2 bases or up to 3 bases) in the sequence of the molecular barcode portion (indel) The present invention is carried out by classifying nucleic acid molecule groups having sequences sequenced as having C.) into the same cluster.
When this aspect of the second embodiment is combined with the first embodiment, it can be implemented in the step (E) above.

In the second embodiment of the present invention, for example, in digital quantification of a target nucleic acid molecule using a barcode sequence, the obtained nucleic acid sequence is indexed, based on sequence similarity, a barcode and / or a target nucleic acid molecule Can be grouped (clustered). In an aspect of the second embodiment of the invention, the clustering may for example be an insertion or deletion (indel up to 1 base, up to 2 bases or up to 3 bases) in the sequence of the molecular barcode portion (indel) The present invention is performed on a group of nucleic acid molecules obtained excluding the sequences sequenced as having.
When this aspect of the second embodiment is combined with the first embodiment, it can be implemented in the step (E) above.

Also, for example, in one aspect of the second embodiment, nucleic acid sequences can be selected according to whether the sequences of a particular barcode are similar, and groups can be created from the selected sequences. Here, “similar” means that the sequences differ by one, two, three or more bases (eg, insertion, deletion or substitution), but the other bases are identical. The proportion of matching bases between similar base sequences is, for example, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more , 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

Third Embodiment of the Invention In the digital quantification of nucleic acid molecules of interest using barcode sequences, insertions or deletions (indels) may occur in the resulting nucleic acid sequences. In a third embodiment of the invention, in the detection of indels that may occur to a nucleic acid sequence (in particular a barcode sequence), one or more (eg one) arranged in all barcode sequences linked to the nucleic acid molecule Whether part (one or more) or all of the two, three, four, five or six or more fixed bases are changed to a base other than the predetermined fixed base in the original position Can be detected. Also in the third embodiment of the present invention, in the detection of indels that may occur to a nucleic acid sequence (in particular a barcode sequence), one or more (for example one) arranged in all barcode sequences linked to the nucleic acid molecule. Relative to the position of each of one, two, three, four, five, six or more fixed bases and the position of each one or more fixed bases in the sequence of the barcode sequence portion that has been sequenced It may further include specifying by comparing the target positions {eg, each fixed base is usually designed to be any one base selected from the group consisting of A, T, G and C. Or each fixed base may be a combination of A and T, a combination of A and G, a combination of A and C, a combination of T and G, a combination of T and C, a combination of G and C, A and T And G combinations, A, T and C Can be designed to be a base selected from the bases contained in any one combination selected from the group consisting of: a combination of A, G and C, and a combination of T, G and C} . This makes it possible to use indel as an indicator that one or more fixed bases are present at a position deviated from the predetermined position, and preferably, as a further index that another base is present at the position where the fixed base should be present. Can be detected. For example, if one or more, for example, two or more fixed bases are present at positions deviated from the predetermined position by the same number of bases, it can be determined that indel is detected. When indel is detected, a nucleic acid molecule group having a sequence sequenced as having indel may be classified into the same cluster as a sequence having no indel, or a nucleic acid molecule group having a sequence sequenced as having indel (Eg, nucleic acid molecules having a sequence sequenced as having indel may be excluded from the obtained sequence information, or nucleic acid molecules having a sequence sequenced as having indel Clusters of nucleic acid molecules may be excluded. In this aspect, when two or more fixed bases are present, the fixed bases may preferably have one or more other bases intervened between the fixed bases. Here, "fixed base" refers to a common position which is present at a predetermined position from the end (5 'end, or 3' end, or 5 'end and 3' end) of a barcode sequence in a plurality of barcode sequences. (Here, the common base may be a base determined by design common to a plurality of barcode sequences as described above).
When this aspect of the third embodiment is combined with the first embodiment, it can be implemented in the step (E). This aspect of the third embodiment can be implemented in the detection of indel when combined with the second embodiment.

Further, according to the first embodiment of the present invention,
Method for analysis of nucleic acids:
(I) subjecting a mixture of a plurality of target nucleic acid molecules to which a molecular barcode and an index have been added to sequencing to obtain sequence information;
(II) From the sequence information obtained in (I) above, a sequence having a specific index or a sequence similar thereto, and / or a sequence having a specific molecular barcode or a sequence analogous thereto is selected and selected Creating a group by the arranged array;
(III) determining, in the group created in (II) above, the pair of the index with the highest detection frequency and the molecular barcode as the correct pair of the index and the molecular barcode;
May be included.

Furthermore, a second embodiment of the present invention is a method of analyzing nucleic acid:
(I) subjecting a mixture of a plurality of nucleic acid molecules to which molecular barcodes have been added to sequencing to obtain sequence information;
(II) selecting a sequence having a specific molecular barcode or a sequence similar thereto from the sequence information obtained in (I) above, and creating a group from the selected sequence;
May be included.

Furthermore, a third embodiment of the present invention is a method of analyzing nucleic acid:
(I) subjecting a mixture of a plurality of nucleic acid molecules to which a molecular barcode having a fixed base at a specific position is added to sequencing to obtain sequence information;
(IIa) excluding from analysis a sequence having a molecular barcode which does not contain the fixed base at the specific position;
May be included.

In each of the above first, second and third embodiments, the target nucleic acid molecule to which at least a molecular barcode has been added may be subjected to amplification prior to step (I). Here, at least a target nucleic acid molecule to which a molecular barcode has been added means that an index may be further added if at least a molecular barcode is added, and an index may not be added. Do.

In each of the above first, second and third embodiments, the molecular barcode is a known method, eg, when amplifying a nucleic acid molecule of interest using a primer comprising the molecular barcode sequence (eg It can be attached to the target nucleic acid molecule by chain reaction).
In each of the first, second and third embodiments, an index may be added to the amplification product of the target nucleic acid molecule to which the molecular barcode has been added. Methods for adding an index to an amplification product include known methods, for example, adapter ligation using an adapter having an index sequence.
In each of the first, second and third embodiments, an index may be added to the target nucleic acid molecule along with the molecular barcode. For example, as a method of adding an index and a molecular barcode to a target nucleic acid molecule, the target nucleic acid molecule is amplified (eg, polymerase chain reaction) using a known method The method is mentioned.

The method of the first embodiment can be implemented in combination with the second embodiment. For example, in each of the first and second embodiments, the sequence similar to the sequence having the specific molecular barcode in step (II) is less than the predetermined number of bases and the sequence having the specific molecular barcode. Or a sequence containing a mismatched base in the molecular barcode sequence portion. Here, the predetermined number of bases is an integer in the range of 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. Or 0, 1, 2, or 3. In a sequence including a mismatched base not more than a predetermined number of bases in the molecular barcode sequence portion, the base other than the mismatched base exactly matches the sequence of the specific molecular barcode.

The method of the first embodiment can be implemented in combination with the third embodiment. The method of the second embodiment can be implemented in combination with the third embodiment.
For example, in each of the first and second embodiments, the molecular barcode may have a fixed base at a particular position.
In each of the first and second embodiments, a sequence similar to the sequence with the specific molecular barcode in step (II) comprises the fixed base at the specific position, and / or the fixed base May be selected based on the fact that the position of is shifted from the specific position.
In each of the first and second embodiments, the method may further include excluding, from analysis, a sequence having a molecular barcode that does not include the fixed base at the specific position. For example, in this embodiment, in the case of clustering molecular barcodes with Distance = 0 or when clustering with Distance = 1 or more, a sequence having molecular barcodes that do not contain the fixed base at the specific position is analyzed It may further include excluding. In this case, excluding from the analysis a sequence having a molecular barcode that does not contain the fixed base at the specific position may be before, after or during clustering.
In each of the first and second embodiments, a sequence having a molecular barcode which does not contain the fixed base at the specific position may be excluded from the sequence information of step (I), which is prepared in step (II) It may be excluded from the analyzed group.
Alternatively, in each of the first, second and third embodiments, sequence information consisting of a sequence comprising the fixed base at the specific position in step (I) or after step (I) You may get it. Alternatively, in the first embodiment, in step (II) or after step (II), a group consisting of a sequence comprising the fixed base at the specific position may be obtained. That is, in the method of analyzing a nucleic acid according to the third embodiment, in place of step (IIa), step (IIb): in the step (I), or after step (I), the identification in the molecular barcode portion Step (II): a sequence having a specific molecular barcode or similar thereto from step (II): the sequence information obtained in (I) above Select sequences to create a group according to the selected sequences, and step (IIc): in step (II) or after step (II), at the specific position in the molecular barcode portion You may obtain the group which consists of a sequence containing the said fixed base. The sequence information or group consisting of the sequence containing the fixed base at the specific position in the molecular barcode portion may consist of the sequence containing the fixed base at all specific positions. The sequence information or group consisting of a sequence including the fixed base at the specific position in the molecular barcode portion is n when the number of fixed bases is n (wherein n is a natural number), Alternatively, it may be composed of a sequence including fixed bases at a specific position of nm (where m can be a natural number ranging from 1, 2, 3 or 1 to n-1).

In each of the first, second and third embodiments, in step (III), the index and molecular barcode pair other than the determined correct pair are determined as the index and the molecular barcode mispair Also, the determined mispair may be excluded from the analysis.

In each of the first, second and third embodiments, the nucleic acid analysis method is based on the number of groups created by the sequence having the specific molecular barcode or the sequence similar thereto. The method may further comprise the step of determining the number of target nucleic acid molecules contained in the sample from which the molecule is derived.

Those skilled in the art can understand that the first embodiment of the present invention, the second embodiment of the present invention, and the third embodiment of the present invention can be implemented in free combination. For example, the first embodiment of the present invention can be combined with the second embodiment of the present invention, and the first embodiment of the present invention can be combined with the third embodiment of the present invention. . The first embodiment of the present invention may be combined with the second and third embodiments of the present invention. Furthermore, the second embodiment of the present invention can combine the third embodiment of the present invention.

Fourth Embodiment of the Present Invention A fourth embodiment of the present invention is a method for digitally quantifying a target nucleic acid molecule using a barcode sequence, comprising: And a third embodiment, and a method comprising the implementation of an embodiment selected from the group consisting of these combinations.

A fourth embodiment of the present invention is a method for digital quantification of a nucleic acid molecule of interest using a barcoded sequence comprising:
(E) selecting a nucleic acid molecule containing the sequence of the target nucleic acid molecule from the obtained sequence information;
(F) clustering the nucleic acid molecules selected in (e) above for each unique molecular barcode sequence, and then identifying a cluster having a plurality of sequences in the index nucleic acid molecule portion,
(G) In each of the clusters identified in (f), the index with the highest detection frequency and the molecular barcode pair are identified as the correctly indexed target nucleic acid molecules, and the other index and molecular barcode pairs are identified. Determining that it is a mispair, and
{Wherein it may further include determining that the index is incorrect in Mispair},
The purpose contained in the sample corresponding to the index based on the number of sequence types of the unique molecular barcode linked to the correctly indexed target nucleic acid molecule (or the number of clusters of the target nucleic acid molecule correctly indexed) Determine the number of nucleic acid molecules,
It may be a method. Here, in one embodiment, in step (g), the number of kinds of unique molecular barcode sequences linked to the correctly indexed target nucleic acid molecule (or the number of clusters of the target nucleic acid molecule correctly indexed) The number may be determined as the number of target nucleic acid molecules contained in the sample corresponding to the index, and it is considered that the accuracy of quantification increases in principle as the number of reads increases.

The fourth embodiment of the present invention is
(A) separately obtaining a plurality of samples comprising a nucleic acid molecule (eg, DNA or RNA) and {at least one of the samples comprising the nucleic acid molecule of interest},
(B) A target nucleic acid molecule in which an arbitrary molecular barcode is linked to each of the target nucleic acid molecules in each of a plurality of obtained samples before amplification of a nucleic acid molecule contained in the sample, and different molecular barcodes are linked to each other Obtaining
(C) Before mixing a plurality of samples, a unique index is added to the target nucleic acid molecule for each sample containing a plurality of target nucleic acid molecules, and a library of target nucleic acid molecules in which different indexes are linked for each sample derived (The order of step B and step C may be earlier; and after step (b) or (c), the nucleic acid molecule can be amplified to obtain an amplification product of the target nucleic acid molecule),
(D) in a mixture containing the amplification products of the nucleic acid molecule obtained after (b) and (c) above (the sample is mixed after step (c) and after the sample is mixed, step (b) ) May be performed, or all the samples may be mixed after performing step (b), and it is after step (b) that the amplification product of the nucleic acid molecule to which the molecular barcode is linked is obtained. The sample may be mixed before obtaining the amplification product, and after obtaining the amplification product, the sample containing the amplification product may be mixed), an index unique to each sample, and a unique or optional for each target nucleic acid molecule And sequencing the nucleic acid molecules to which the molecular barcodes have been added to identify the sequence of the index portion, the sequence of the molecular barcode portion, and the sequence of the nucleic acid molecule portion linked thereto for each nucleic acid molecule. It is also good.

In the fourth embodiment of the present invention, for example, as described in the second embodiment, in the (f), the clustering is
(I) In the sequence of the molecular barcode portion, by grouping nucleic acid molecule groups having the same sequence as the unique molecular barcode sequence into the same cluster;
(Ii) in the sequence of the molecular barcode portion, by grouping nucleic acid molecule groups having a unique molecular barcode sequence and a sequence having up to one mismatch into the same cluster;
(Iii) in the sequence of the molecular barcode portion, by grouping nucleic acid molecules having a unique molecular barcode sequence and a sequence having up to 2 bases of mismatch into the same cluster; or (iv) the molecular barcode In the partial sequence, it may be performed by classifying nucleic acid molecule groups having a sequence of unique molecular barcodes and a sequence having up to 3 bases into the same cluster.

In the fourth embodiment of the present invention, for example, as described in the second embodiment, in (e), clustering is
In the sequence of the molecular barcode portion, nucleic acid molecules having a sequence sequenced as having an insertion or deletion (indel) of bases (for example, up to 1 base, up to 2 bases, or up to 3 bases) in the same cluster It may be performed by classification. At this time, the molecular barcode having the fixed base described in the third embodiment may be used.

In the fourth embodiment of the present invention, for example, as described in the second embodiment, in (e), clustering is
A nucleic acid molecule obtained by excluding sequences sequenced as having insertions or deletions (indel) of bases (eg, up to 1 base, up to 2 bases, or up to 3 bases) in the sequence of the molecular barcode portion It may be performed on a group. At this time, the molecular barcode having the fixed base described in the third embodiment may be used.

In this way, in digital quantification, errors in nucleic acid sequences that may occur can be corrected and the accuracy of digital quantification can be improved. That is, according to the present invention, using a sufficiently large number of molecular barcodes as compared to the number of original target nucleic acid molecules in the sample, each of the target nucleic acid molecules can be Accurate digital quantification is achieved by labeling and obtaining a sufficiently large number of leads compared to the number of original target nucleic acid molecules and detecting all the molecular barcodes attached to each target nucleic acid molecule. It will be possible.

In the modern big data era of biology, accurate quantification of biomolecules in system-wide measurements is required. Because the quality of the analysis is highly dependent on the initial raw data. For this reason, digital quantification of nucleic acid molecules using DNA tags (designated as “primer ID” ¹ , “UMI (unique molecular identifier)” or “molecular barcode”) has been It is being developed. This technology includes gene expression analysis (RNA-Seq) ^2-7 by RNA sequencing, iCLIP (individual-nucleotide resolution UV cross-linking and immunoprecipitation) ⁸ , antibody repertoire analysis ⁹ , bacterial 16S rRNA gene analysis ¹⁰ , ¹¹ , and ChIP It is used for many applications in next generation sequencing platforms such as -nexus (chromatin immunoprecipitation experiments with nucleotide resolution through exonuclease, unique barcode and single ligation) ¹² . These methods make it possible to digitally accurately determine the absolute number of molecules in a given sample, even in the presence of noise and / or bias in the measurement system. RNA-Seq using molecular barcodes, ie digital RNA-Seq (dRNA-Seq) ³ or quantitative RNA-Seq ¹³ is one of the most widely used applications of digital counting. dRNA-Seq is often used for single cell gene expression analysis as it works well even for small sample sizes. The detection limit is important in such measurement. Because, single cells have been shown to have many low copy RNAs ^13,14 , and detection limits indicate that there are many potentially undetected low copy RNAs, which are biological Because it can affect the subsequent interpretation of the phenomenon. Therefore, since the barcode system used determines the detection limit of nucleic acid quantification, the investigation of the efficacy of barcodes for absolute and digital quantification is crucial. In addition, the simultaneous effectiveness of the bar code's ability to count high copy numbers is also important. For example, random base barcodes can be used to label thousands of viral RNAs ¹ , and high-throughput single cell RNA-Seq studies, where barcodes are individual cells in a single sequencing run Because it can be used to identify thousands of cells in ⁷⁾ .
The general procedure for digital quantification of nucleic acid molecules is as follows (see FIG. 1, panel A). (I) Uniquely tag each RNA (or complementary DNA or cDNA) or DNA with externally added DNA (molecular barcodes) containing diverse sequences ^1-3 . (Ii) Amplify the barcoded DNA or cDNA (produced from RNA when starting from RNA). (Iii) Both the target nucleic acid sequence of the barcoded and amplified (c) DNA and the barcoded sequence are tandemly sequenced. (Iv) As theoretically proposed ¹⁵ , each target nucleic acid (or gene) to give an absolute copy number of the original target nucleic acid (that is, RNA before amplification or (c) DNA) before amplification. ), The number of unique barcodes is quantified rather than the number of amplified molecules (the so-called "read number"). This scheme can eliminate the effects of noise and / or bias generated at various steps during measurement of the system (eg, from amplification, sequencing and / or analysis). In order to ensure that the digital counting system works properly, it is ensured (or nearly guaranteed) that each target nucleic acid molecule is uniquely tagged and the measured number of unique molecular barcodes is A variety of barcode sequences must be used to equal the number of target nucleic acid molecules of interest16, ¹⁷ (first requirement below). Also, it is empirically considered that sufficient sequence depth is required for accurate counting ^{18, 19} (second requirement below).
The following two types of barcode designs are typically used in digital counting schemes: sequence limited barcodes (each barcode sequence is designed separately) and non-sequence limited barcodes ("random" Base) sometimes referred to as a barcode). When sequence limiting barcodes were previously used, the diversity of barcode sequences required for accurate quantitation was estimated by theoretical calculations ¹⁶ and for absolute quantitation of barcoded molecules. The capacity of this technology was experimentally confirmed ^3,16 . However, the use of sequence limiting barcodes has the following disadvantages: Many different individually designed barcode sequences have to be prepared for the measurement of high dynamic range, which is It is not cost effective. In order to minimize costs while increasing the dynamic range of counting, random (or pseudorandom) base barcodes are used instead ^{2,4-9,11,12,18,20} . Again, it should be determined that the sequence diversity of the barcode set is sufficient ^{17, 18} . However, unlike sequence-limited barcodes, sequence changes in barcodes due to sequencing and / or amplification errors (barcode sequences newly generated from one of these errors may become false positives) ²¹ For this reason, this survey is not a trivial matter. That is, errors can cause an overestimation of the number of molecules in the sample (in the case of sequence limited barcodes, all used barcode sequences are known, which is also known as all unused barcode sequences) Note that it means that sequences resulting from errors can be identified and excluded). This problem is approached by using computer analysis to rule out errors based on the rational assumption that similar barcode sequences occur through errors originating from the same original barcode sequence. Furthermore, Sudbery et al. Recently showed the effectiveness of random base UMI (molecular barcodes) based on computer analysis by modeling errors on a limited dynamic range (up to 100 molecules) ²² . However, the effectiveness of random base molecule barcodes for accurate digital counting is based on experiments that may explicitly include effects not present in the theoretical model, especially with a quantitative meaning of ^{7, 20} and high dynamic range Was not clearly stated.
Here, we use random base molecule barcodes for digital quantitation of absolute numbers of barcoded DNA molecules when using specific barcode designs and after computer analysis We show experimentally what we can do. In order to investigate the effectiveness of the barcode itself by excluding other effects such as barcode addition and / or reverse transcription which may vary in different applications, we have DNA molecules comprising barcode sequences Was synthesized and quantified by sequencing on the amplified molecule (see the dotted box in panel A of FIG. 1). We have investigated the above two requirements quantitatively for accurate digital counts; (i) using a sufficiently large set of barcode sequences compared to the number of given molecules (above (As panel B in FIG. 1), and (ii) that sufficient sequencing depth is achieved as compared to the number of predetermined molecules (panel C in FIG. 1). We then show experimentally that both the number of molecular inputs and the number of measured molecular outputs are consistent through a model measurement system that meets the two requirements. To meet these two requirements, ie to ensure that the digital counting system works, we introduce fixed bases into the random barcode sequence for error detection and in-house Perform barcode sequence clustering using the developed software, and make use of the information from molecular barcodes to target nucleic acid sequences (templates) in the cross contamination and mapping process between differently indexed samples Misidentification was identified and excluded. The present results show that accurate quantification of barcoded nucleic acid molecules in any given sample is high, through proper barcode design (including minimal required barcode length) and sufficient sequencing depth It shows that it can be achieved in the dynamic range (from 1 to over 10 ⁴ and potentially up to 10 ¹⁵ molecules).
Hereinafter, in this example, the term "random" is used. In this example, this term is randomly synthesized by an experimenter in order to ensure great diversity in the sequence without designing the sequence. It means that.
[Method]
Library Preparation Single stranded DNA templates containing random bases were purchased from Integrated DNA Technologies, Inc., Coralville, IA, USA (see FIG. 13). The concentration of each template is at 260 nm using a spectrophotometer (NanoDrop 1000; Thermo Fisher Scientific Inc., Mass., USA) with the absorption coefficient described in the provided specification sheet (Integrated DNA Technologies, Inc.) It was measured by the absorption of Template DNA was stored at −30 ° C. at 50 μM in 0.1% (v / v) TWEEN 20 (Sigma-Aldrich, St. Louis, Mo., USA) solution. To control the concentration of DNA template for amplification, all templates were diluted with water (distilled water, deionized, sterile, NIPPON GENE CO., LTD., Toyama, Japan) and 0.1% TWEEN 20, The final copy number was mixed in the PCR tube. The amplification was carried out by PCR using MightyAmp (TAKARA BIO INC., Shiga, Japan) using 0.3 μM of each primer (see FIG. 14) in 25 μL samples. Two tubes were prepared independently from 50 μM template stock and distinguished by the designed index in one of the primers (see FIG. 14). Thermal cycling (ProFlex PCR system; Themo Fisher Scientific Inc.) was performed as follows: half cycle at 98 ° C .; 10 seconds at 98 ° C., 10 seconds at 60 ° C., and 1 minute at 68 ° C. 4 cycles; 19 cycles of 98 ° C. for 10 seconds, 60 ° C. for 2 seconds, and 68 ° C. for 1 minute; 68 cycles of 1⁄5 of the cycle; Then, the amplified products were twice column purification ^{(DNA Clean & Concentrator TM -5;} Zymo Research Corp, CA, USA) , and the length distribution of the amplified product 2100 Bioanalyzer (Agilent Technologies, Inc., CA, USA) and It confirmed using. Concentrations were determined by qPCR kit (KK4602; KAPA Biosystems, Inc., MA, USA) using a real-time PCR system (7500; Themo Fisher Scientific Inc.).

Sequencing index added two samples (CGCTCATT: index A (index A), GAGATTCC: index B (index B)) for 150 cycle kit v3 (Read 1: 100 cycles, Read 2: 50 cycles, Index 1: 8 Single-run sequencing using MiSeq sequencer (Illumina, Inc.). Read 2 was not used for analysis because the sequence in Read 2 is part of the sequence in Read 1. Raw sequence data used for analysis was deposited at GEO database GSE94895.

Analysis

Read 1 sequences were sorted by index A and B and fastq files for each index were generated using MiSeq. In some cases, 100%, 32%, 10%, 3.2%, 1%, 0.32%, and 0.1% of the leads were randomly sampled. MiSeq fastq files were filtered by sequence length (≧ 34 bp and ≦ 39 bp for short templates and 90 90 bp for long templates). Lead alignment to target nucleic acids sequences using the nucleic acid sequence of interest of eleven template as a reference (see "target" in Fig. 13), longer templates using Bowtie2 v.2.2.9 ²⁷ (LT) and short templates ( Implemented separately for ST). Basically, uniquely mapped reads were used for the next analysis. The barcode region was 50 bp from the 5 'end for the long template and 30 bp from the 5' end for the short template (see "barcode" in FIG. 13) and these were extracted from the mapped reads. The fixed bases in the barcode region (up to 6 bases for short templates and up to 12 bases for long templates; see Figure 13 "barcode") are used for filtering and at least one fixed base mismatch I have excluded the barcode I have. The in-house software Nucleotide Sequence Clusterizer was then used to cluster barcodes at Distance = 0, 1, 2, or 3. The number of clusters was considered to be the number of molecules before amplification. The leads with index A and B were integrated prior to clustering when cross-contamination of the index was considered. In the latter, multiply mapped reads were also used for further analysis. And, after clustering, if there were clusters containing multiple indexes, we excluded minority leads. If the number of reads of index A and index B was the same, a coefficient of 0.5 was given to both index A and index B. Similarly, if misalignment was also taken into consideration, all reads mapped to the template with index A and index B were integrated before clustering. When one lead was mapped to multiple templates, a factor of 1 / (number of different templates) was given for each template. After clustering, minority leads were excluded if there were clusters containing leads and / or indexes mapped to multiple target nucleic acids. If the number of reads and / or indices mapped to different templates is the same, a factor of 1 / (number of different templates and / or indices) for each of the nucleic acids of interest and / or indices mapped to multiple Gave. The number of leads in each process is as shown in FIG.

Nucleotide Sequence Clusterizer
The in-house software named "Nucleotide Sequence Clusterizer" was coded in C for clustering. This tool performs clustering of DNA sequences using the identified nucleotide positions of each sequence. This tool performs bounded single link clustering: First, each sequence is in its own cluster. If any two sequences differ from each other by no more than D mismatches, their clusters were integrated together. Here, D is a configurable "Distance" parameter. This process continues until there are no more clusters to consolidate, at which point the Nucleotide Sequence Clusterizer reports the number of clusters and the sequences within each cluster. The Nucleotide Sequence Clusterizer is available upon request.

In this example, it was examined whether the digital count system of nucleic acid using random base barcode can accurately measure the absolute number of DNA molecules contained in a sample. As shown in FIG. 13, two kinds of template DNAs were designed: six long templates (LT1 to 6) and five short templates (ST1 to 5).

As shown in FIG. 13, the LT1-6 nucleic acid molecules are directed from the 5 'end to the 3' end side,
It was designed to be the sequence of SEQ ID NO: 1-barcode sequence-target nucleic acid sequence-the sequence of SEQ ID NO: 2. The barcode sequences of LT1-6 and the target nucleic acid sequences are shown in SEQ ID NOs: 5-16.
Further, as shown in FIG. 13, the nucleic acid molecules of ST1 to 5 are directed from the 5 'end to the 3' end side,
It was designed to be the sequence of SEQ ID NO: 3-barcode sequence-target nucleic acid sequence-the sequence of SEQ ID NO: 4. The barcode sequences of ST1-5 and the target nucleic acid sequences are shown in SEQ ID NOs: 17-26.

These template DNAs all contain random base barcodes, shown as molecular barcodes in panel A of FIG. 1, and the long template is downstream of a 50 base barcode consisting of 38 random bases and 12 fixed bases. The short template had the target nucleic acid sequence of 8 bases downstream of the barcode of 30 bases consisting of 24 random bases and 6 fixed bases (FIG. 13). reference). Also, all templates contained common sequences at both the 5 'and 3' ends used for PCR amplification (see Figures 13 and 14). In this example, 40 000, 40000, 4000, 300, 100 and 20 copies of LT1, LT2, LT3, LT4, LT5 and LT6, 20000 copies of ST1 and ST2, and 4000 copies, respectively, as model measurement samples. Two identical samples were prepared, each containing ST3, ST4 and ST5. Amplify these templates in these two samples distinguished by two different indexes (Index A and Index B) and sequence the amplification products using MiSeq, read 11, 992, 843 for Index A, Index B In contrast, 15,373, 718 leads were obtained (see FIG. 15).

In this example, the sequences of index A and B were added to the template by including in the reverse primer for amplification (see FIG. 14).
Sequence of reverse primer for amplification of index A (Rv primer in FIG. 14):
CAAGCAGAGAGACGGCATACGAGAT AATGAGCG GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (SEQ ID NO: 28)
Sequence of reverse primer for amplification of index B (Rv primer 2 in FIG. 14):
CAAGCAGAGACGCGATACGAGAT GGA ATCTC GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (SEQ ID NO: 29)
The underlined portion in the nucleic acid sequence of SEQ ID NO: 28 corresponds to the nucleic acid sequence of index A, and the underlined portion in the nucleic acid sequence of SEQ ID NO: 29 corresponds to the nucleic acid sequence of index B.

Then, all reads are sorted by MiSeq for each index (A and B), the sorted reads are mapped to the reference consisting of the target nucleic acid sequence, and the number of reads sequenced (ie, the number of amplified molecules) Instead of counting the number of molecules for each index and template was quantified digitally by counting the number of unique barcodes (or the number of barcode clusters).

Second, the two requirements for accurate digital quantitation in the presence of errors (ie how many random bases are needed in a barcode to count a given number of molecules in a sample, and per molecule The number of leads (defined as the “coverage”) was examined (FIGS. 2 and 8). Regarding the first requirement, each sorted index and each random index in each template is changed on the computer (4 to 38 bases for LT and 4 to 24 bases for ST) The unique barcode number for the mold was determined (Figure 2 panel A and Figure 8A; gray line). The number of unique barcodes determined increased dramatically as the number of random bases in the barcodes was increased. This implies that a certain minimum number of random bases is required to quantify a given number of molecules. Even if the number of possible barcode sequences is artificially increased (by increasing the length of the barcode), the number of original target nucleic acid sequences to be measured can not exceed 20000 of the original copy number. The plateau is expected to be at 20000 because it should not increase. However, the expected plateau was not observed in the region of larger number of random bases, and the number of unique barcodes determined increased monotonically as the number of random bases increased. For the second requirement, change the coverage of the sequencing on a computer by randomly excluding some of the reads, and the number of unique barcodes with the remaining reads for each index and template Were determined (panel C in FIG. 2 and panel C in FIG. 8; gray line). If the scheme of digital counting is working, the plateau should be determined because the number of unique barcodes to be identified should not depend on the coverage (sequence depth) if the coverage reaches a sufficient level Will be observed in the plot of Even if the sequence depth (ie, the number of times each barcode is read) is increased, the measured number of original target nucleic acid sequences should not increase beyond 20000 of the original copy number, so the plateau is 20000. Expected to be. However, this expected plateau was not observed, and the number of unique barcodes determined increased monotonically as coverage increased. This implies that the digital counting system needs improvement under this condition.

The reason why the plateau was not observed in these figures (panel A and panel C in FIG. 2 and panel A and panel C in FIG. 8) was finally sequenced as compared to the actual barcode sequence input In the output of the barcode, it can be explained by base changes, such as substitution errors and insertion-deletion (indel) errors. The barcoded sequences were clustered using the in-house software Nucleotide Sequence Clusterizer to eliminate substitution errors (probably due to sequence errors and / or polymerase amplification errors). In the clustering procedure, a parameter called "Distance" was introduced: where Distance indicates the number of bases that differ between two given barcode sequences. For example, if one barcode sequence is exactly the same as another barcode sequence except for two base changes at any two positions, then the Distance between these two barcode sequences is 2. Therefore, after clustering with Distance = 2 as a parameter, all barcode sequences in a given cluster are within Distance = 2 from at least one other barcode sequence in that cluster (included in the cluster Any molecule is not necessarily within Distance = 2 from all other sequences). In essence, the original analysis to count the number of unique barcodes without clustering can be said to have been performed by clustering at Distance = 0. The number of barcode clusters was determined with Distance = 0, 1, 2 or 3 (see panel A in Figure 3 and panel A in Figure 9). If there were enough kinds of barcodes for a given number of molecules, the number of barcode clusters would be expected to approach a constant value as Distance increased, and this trend was actually observed. Determine the number of barcode clusters determined by using the clustering with the longest Distance (Distance = 3) performed to identify the effect of clustering on the two requirements for accurate digital quantification. Plotted as a function of number (see panel A of FIG. 2 and blue line of panel A of FIG. 8) and coverage (see panel C of FIG. 2 and blue line of panel C of FIG. 8). More plateau-like curves were observed for both plots, but the number of barcode clusters determined was still monotonically increasing, particularly as coverage was increased.

Next, an attempt was made to exclude the influence of errors of the insertion-deletion (indel) type by excluding the reads containing mismatched bases at the fixed base position of the barcode sequence among the sequenced reads (FIG. 13). reference). If the barcode sequence output contained a mismatched base at any of these fixed base positions, the remaining base offset from the designated "reading frame" defined by the fixed base position An insertion or deletion of the base at another position in the barcode sequence was found to occur. In order to investigate the effect of this process on the digital counting system, the positional dependence of fixed bases in the barcode sequence was examined. The number of barcode clusters was determined when using one fixed base for this exclusion procedure (see panel B of FIG. 3 and panel B of FIG. 9). The number of barcode clusters decreased as the position of the fixed base moved away from the sequencing primer site. This is reasonable as fixed base mismatches can detect indel-type sequence changes that occur between the sequence initiation site and the position of the fixed base. Also, the dependence of the number of fixed bases on the number of barcode clusters determined was analyzed. At this time, the fixed base located at the farthest position from the sequence primer site was used (see panel C of FIG. 3 and panel C of FIG. 9). When the number of fixed bases used was small, the number of barcode clusters determined decreased significantly, and as the number of fixed bases used increased, the number of barcode clusters determined became almost constant. To observe the effect of mismatch exclusion on the above two requirements for accurate digital quantification, the number of random bases (panel A in FIG. 2 and panel A in FIG. 8; green line) and coverage (FIG. 2) The number of barcode clusters determined was plotted as a function of panel C and panel C of FIG. 8; green line). The mismatch exclusion process was performed using the highest number of fixed bases used (6 bases for short templates, 12 bases for long templates). As a result, a plateau-like curve is seen for any of the plots (panels A and C in FIG. 2 and panels A and C in FIG. 8), which indicates that indel error exclusion with fixed bases causes digital quantification. Indicates that you have made it more accurate.

As another problem, it was found that cross contamination between samples occurred. This is believed to cause a slight increase in the number of clusters observed in the plateau-like phase in the green lines of panel C of FIG. 2 and panel C of FIG. Two samples were each labeled with different indexes (index A and index B) by amplification primers during PCR, and two samples amplified by PCR in two separate tubes were simultaneously sequenced. When barcodes were clustered using both Index A and Index B, a small fraction of barcode clusters containing both indexes was found. This is also reported by Jaitin et al. ⁵ This may have occurred without cross contamination since the barcoded template for PCR amplification was randomly selected from the original template pool. However, even in the case of a short template, the type of barcode sequence is theoretically 2.8 × 10 ¹⁴ (= 4 ²⁴ ), so that two original templates having completely the same barcode are amplified. The possibility of being added to the tube is considered to be very small. Therefore, it was considered that either the PCR primer containing the specific index was mixed in the tube, the index sequence had an error, and / or index switching occurred in the sequencing step (Sinha , R. et al., Biorxiv, 10.1101 / 125724 (2017)). To eliminate this effect, first, all the leads sorted into two indices for each template were mixed and clustering was performed on these mixed leads. If multiple indexes (in this case, two indexes) were found in one barcode cluster, the barcode cluster was counted as having an index including the highest number of reads among the sequenced reads. Using this process, it was finally found that the determined number of clusters showed a plateau as a function of coverage (see panel C of FIG. 2 and yellow lines of panel C of FIG. 8). Importantly, the blue lines in panel C of FIG. 2 and panel C of FIG. 8 show that the number of clusters determined to increase in coverage slightly increases, but the effect of index switching is With the above process excluded, the number of clusters showed a plateau even if the coverage rate increased.

Since the same barcode sequence is considered not to be used for both indexes, it was determined for the sum of indexes A and B to confirm the first requirement for accurate digital quantification The number of clusters was plotted (see the yellow lines in panel B of FIG. 2 and panel B of FIG. 8). Since there was still a plateau, the number of random bases used was within the acceptable range for performing accurate digital quantification.

The above example may occur when mixing and analyzing a plurality of samples, "index switching" affects the accuracy of barcode clustering, and exclusion of index switching (miss index) It is shown that the process allows for a digital quantitative system with improved accuracy and accuracy not affected by coverage.

As cross contamination between samples was found, we then examined for misidentification in the process of mapping leads to reference. We followed the same process as we did for the index issue. Here, all the leads sorted to two indices and mapped to either template were mixed and then clustering was performed on the mixed leads. Next, when multiple templates and / or multiple indexes were found in one barcode cluster, barcode clusters for the template and index that showed the highest number of reads among the sequenced reads were counted. However, no significant difference was observed in the number of clusters determined as a function of coverage throughout this process (see panel C in FIG. 2 and dotted red in panel C in FIG. 8). This suggested that in this system misidentification does not occur so frequently. Index A, Index B and all to confirm the first requirement for accurate digital quantification, as it is assumed that the same barcode sequence will not be used for both index and all templates The number of clusters determined was plotted for the sum of the molds (see red dotted line in FIG. 2 panel B and FIG. 8 panel B). Because there were still plateaus, the number of random bases used was within the acceptable range for performing accurate digital counts, even when explaining the misidentification of the template. In this example, the effect of misidentification was less, but this process becomes important in analyzes where a larger amount of reference is used (eg, RNA-Seq). This is because misidentification can occur more frequently in such an analysis.

To further understand what happened in the above analysis process, a histogram of coverage was created for each process (Figure 10). Histograms that counted the number of unique barcodes (without any treatment above) had large peaks that mainly included low lead clusters. These low lead clusters, as measured by this digital counting method, artificially increase the output copy number of the target nucleic acid sequence (artificially not present in the original sample due to sequence errors, indel errors, etc.) Bar code sequences), which must be excluded in order for the system to make more accurate quantitation (the two requirements above). Although this peak decreased dramatically after the first two processing steps, this suggests that the barcode sequences generated mainly by sequencing errors were excluded by these processing steps.

The above barcode design and computer analysis were shown to meet two of the above requirements for accurate digital counting when using four specific templates (ST1, ST2, LT1 and LT2) (Figure 2 panels AC and FIG. 8 panels AC)). The parameters were then optimized and these analyzes were applied for all templates containing a wide range of copy numbers from 20 to 40,000. Since the number of clusters determined in the case of Distance = 2 as a parameter had already approached a constant value (see panel A of FIG. 3 and panel A of FIG. 9), Distance = 2 was used in the subsequent analysis. As for the number of fixed bases, when the number of fixed bases is 4, the number of clusters to be determined approaches a constant value (see panel C of FIG. 3 and panel C of FIG. 9). The number was 4 (for all templates, the 16th, 21st, 24th and 28th from the left used a barcode (FIG. 13) with fixed bases). Cross contamination of the index and misidentification of the template were also considered. It is believed that all of the above quantitative analysis and insights can be used to accurately quantify the nucleic acid molecule of interest using the digital counting scheme of the present invention. Based on these conditions, two requirements were examined for all the molds to determine the dynamic range of this digital counting system (see panels A, B and 11 of FIG. 4). For coverage dependency, use 20 random bases for clustering (panel A and 11 in Figure 4), and for the dependence on the number of random bases, analyze 10% of the original total number of reads We decided to use (panel B of FIG. 4). Because, based on the previous initial analysis of the four original molds, it was thought that both parameters should still function (panel A in FIG. 2, panel C in FIG. 2, diagram Panel A of FIG. 8 and panel C of FIG. If less than 100% of the leads were used for analysis, the leads were randomly selected and the process repeated eight times to determine the mean and standard deviation (FIGS. 4A-4C and FIG. 11). As shown in FIGS. 4A-4C and FIG. 11, in the plots as a function of number of random bases and coverage, a plateau was present for all templates. This suggested that the chosen parameters allow accurate digital quantification over a wide range of copy number templates.

The determined number of barcodes in panel A of Figure 4 and Figure 11 is included in the sample tube prior to PCR amplification with a coverage of 12.6-20.9 (when 10% of the leads are sampled) Correspond to the number of molds. These values were used to compare the number of input molecules determined by optical density with the number of output molecules determined by the digital counting method of the present invention (see panel C of FIG. 4). As a result, the value of the number of input molecules and the value of the number of output molecules showed a high correlation (Pearson's product ratio correlation coefficient r = 0.990). This output / input ratio is in the range of 0.32-0.45 for the long template (LT) and 0.41-0.57 for the short template (ST) and can be explained by experimental error (e.g. PCR amplification) Statistical error in (at most) 7 stages of template dilution in preparation for This suggests that the digital count scheme based on the parameters presented in this example can quantify the absolute copy number of the nucleic acid molecule prior to PCR amplification.

Based on these results, the required number of random bases can be presented to count the absolute number of molecules in the presence of errors (see FIG. 5, panel A). The x-axis indicates the number of inputs of the molecule to be measured, and the y-axis indicates the number of random bases when each curve in panel B of FIG. 4 and panel B of FIG. Show. Panel B in FIG. 5 shows the dependence of the relative number of clusters on the number of random bases as done in panel B of FIG. Did not have a significant effect). Including these data in panel A of FIG. 5 to show more data in the lower range of a given number of molecules, and, for example, to quantify about 10 ⁵ molecules with an accuracy of over 95% It has been found that at least 16 random bases are required.

Experimentally, it was shown that at most 84,420 molecules (the number of total LTs input) were correctly quantified using 20 random bases (FIG. 4 panel B). This number is considered to be sufficient, for example, to count the number of RNA molecules for individual genes in transcriptome analysis. In practice, the number of molecules that can be measured is limited by the capacity of the MiSeq sequencer.
Based on a simple linear regression on an experimentally measured data set using up to 38 random bases and the number of random bases required depending on the number of molecules given (see Figure 12) It is suggested that about 10 ¹⁵ molecules can be quantified with the measurement system of the present invention. This dynamic range is superior to the current capacity of commercial deep sequencers. Thus, the bottleneck of quantitative analysis with a wide dynamic range is no longer limited by the barcode design, but rather by the throughput of the sequence.

As described above, in the present example, it has been shown that digital counting can be performed to design and use a hybrid molecular barcode including random bases and fixed bases, and the number of molecules in a predetermined sample can be quantified. . Here, appropriate barcode design, sufficient sequence depth, and analysis method with appropriate parameters are used. This makes it possible to measure the number of nucleic acid molecules in a wide, high dynamic range and at low cost. Based on this result, it is possible to suggest the number of random bases and fixed bases needed to count the number of given barcode molecules in the presence of errors (FIG. 5 panel A and FIG. 12). This example also quantitatively demonstrates the additional functional advantages of molecular barcodes. In other words, molecular barcodes are used to identify sample cross contamination (caused by physical contamination of primers, errors in the index, and / or index switching in the sequencing process) or misidentification of the target nucleic acid sequence in the alignment process did. In fact, as mentioned above, the former can solve the serious problems reported in the next generation sequencer platform ^{23, 24} . While the impact of errors may be dependent on the library preparation and / or sequencing platform, the effectiveness of random base barcodes has been demonstrated in general applications and validation of the barcode usage presented here The strategies for are applicable to various platforms. Furthermore, the effectiveness of random base barcodes on barcoded molecules has been demonstrated, so that one skilled in the art, who can vary from application to application, can evaluate the effectiveness or efficacy of barcode addition. The present invention includes gene expression analysis, iCLIP ⁸ , antibody repertoire analysis ⁹ , bacterial 16S rRNA gene analysis ¹⁰ , ¹¹ , count of molecules in ChIP-nexus ¹² as well as cells ⁹ , ²⁵ , ²⁶ , virus ¹ and barcodes Can be widely used in digital counting of nucleic acid quantitation using molecular barcodes, for other applications using Recently, commercially available products such as Single Cell Sequencing Solution (Illumina, Inc., CA, US and Bio-Rad Laboratories, Inc., CA, USA) and Chromium Single Cell 3 'Solution (10x Genomics, Inc. CA, USA) An apparatus may be used to perform these applications. We believe that system biology is promoted based on a large amount of quantitative data obtained experimentally.

Contents of Sequence Listing SEQ ID NO: 1: Base sequence of 5 'region of LT1 to 6 SEQ ID NO: 2: Base sequence of 3' region of LT1 to 6 SEQ ID NO: 3: Base sequence of 5 'region of ST1 to 5 SEQ ID NO: 4: base sequence of 3 'region of ST1 to 5 barcode of SEQ ID NO: 5: LT1 barcode sequence SEQ ID NO: 6: LT1 target nucleic acid sequence SEQ ID NO: 7: LT2 barcode sequence SEQ ID NO: 8: target nucleic acid sequence of LT2 SEQ ID NO: 9: LT3 bar code sequence SEQ ID NO: 10: LT3 target nucleic acid sequence SEQ ID NO: 11: LT4 bar code sequence SEQ ID NO 12: LT4 target nucleic acid sequence SEQ ID NO: 13: LT5 bar code sequence SEQ ID NO 14: LT5 The target nucleic acid sequence SEQ ID NO: 15: LT6 bar code sequence SEQ ID NO 16: LT6 target nucleic acid sequence SEQ ID NO 17: ST1 bar code sequence SEQ ID NO 18: ST1 target nucleic acid sequence SEQ ID NO 19: ST2 barcode sequence SEQ ID NO: 20: ST2 target nucleic acid sequence SEQ ID NO: 21: ST3 barcode sequence SEQ ID NO: 22: ST3 target nucleic acid sequence SEQ ID NO: 23: ST4 barcode sequence SEQ ID NO 24: ST4 purpose Nucleic acid sequence SEQ ID NO: 25: Bar code sequence of ST 5 SEQ ID NO: 26: Target nucleic acid sequence of ST 5 SEQ ID NO: 27: Sequence of forward primer for amplification SEQ ID NO: 28: Sequence of reverse primer for amplification (for index A)
SEQ ID NO: 29: Sequence of reverse primer for amplification (for index B)

References

Claims

Method for analysis of nucleic acids:
(I) subjecting a mixture of a plurality of target nucleic acid molecules to which a molecular barcode and an index have been added to sequencing to obtain sequence information;
(II) From the sequence information obtained in (I) above, a sequence having a specific index or a sequence similar thereto, and / or a sequence having a specific molecular barcode or a sequence analogous thereto is selected and selected Creating a group by the arranged array;
(III) determining, in the group created in (II) above, the pair of the index with the highest detection frequency and the molecular barcode as the correct pair of the index and the molecular barcode;
Method, including.
The method according to claim 1, wherein the nucleic acid of interest at least a molecular barcode has been added is subjected to amplification prior to step (I).
The sequence similar to the sequence having the specific molecular barcode in step (II) is a sequence including a sequence having the specific molecular barcode and a mismatched base having a predetermined number of bases or less in the molecular barcode sequence part. The method of claim 1 or 2.
The method according to any one of claims 1 to 3, wherein the molecular barcode has a fixed base at a specific position.
The sequence similar to the sequence having the specific molecular barcode in step (II) contains the fixed base at the specific position, and / or the position of the fixed base is shifted from the specific position The method according to claim 4, wherein the method is selected on the basis of
5. The method according to claim 4, further comprising excluding from analysis the sequence having a molecular barcode that does not contain the fixed base at the specific position.
In step (III), excluding the index and molecular barcode pairs other than the determined correct pair as the index and molecular barcode mispairing,
A method according to any one of the preceding claims.
Determining the number of target nucleic acid molecules contained in the sample from which the target nucleic acid molecule is derived based on the number of groups generated by the sequence having a specific molecular barcode or a sequence similar thereto. The method according to any one of 1 to 7.
Method for analysis of nucleic acids:
(I) subjecting a mixture of a plurality of nucleic acid molecules to which molecular barcodes have been added to sequencing to obtain sequence information;
(II) selecting a sequence having a specific molecular barcode or a sequence similar thereto from the sequence information obtained in (I) above, and creating a group from the selected sequence;
Method, including.
The sequence similar to the sequence having the specific molecular barcode in step (II) is a sequence including a sequence having the specific molecular barcode and a mismatched base having a predetermined number of bases or less in the molecular barcode sequence part. 10. A method according to item 9.
The method according to claim 9 or 10, wherein the molecular barcode has a fixed base at a specific position.
The sequence similar to the sequence having the specific molecular barcode in step (II) contains the fixed base at the specific position, and / or the position of the fixed base is shifted from the specific position The method according to claim 11, wherein the method is selected on the basis of
The method according to claim 11, further comprising the step of excluding from analysis a sequence having a molecular barcode that does not contain the fixed base at the specific position.
Determining the number of target nucleic acid molecules contained in the sample from which the target nucleic acid molecule is derived based on the number of groups generated by the sequence having a specific molecular barcode or a sequence similar thereto. The method according to any one of 9 to 13.
The method according to any one of claims 9 to 14, wherein the target nucleic acid molecule to which at least a molecular barcode has been added is subjected to amplification prior to step (I).
Method for analysis of nucleic acids:
(I) subjecting a mixture of a plurality of nucleic acid molecules to which a molecular barcode having a fixed base at a specific position is added to sequencing to obtain sequence information;
(IIa) excluding from analysis a sequence having a molecular barcode which does not contain the fixed base at the specific position;
(IIb) obtaining sequence information comprising a sequence comprising the fixed base at the specific position in the step (I) or after the step (I); or (IIc) as the step (II) above Further comprising the step of selecting a sequence having a specific molecular barcode or a sequence similar thereto from the sequence information obtained in 4.), and creating a group according to the selected sequence, and in step (II) or After II) obtaining a group consisting of a sequence comprising the fixed base at the particular position;
Method, including.