WO2019085546A1 - Method for constructing microbial 16s rdna single-molecule level sequencing library - Google Patents

Abstract

Provided is a method for constructing a microbial 16S rDNA single-molecule level sequencing library, relating to the technical field of biochemistry. The library construction method comprises steps of: sample collection and DNA extraction; amplification; purification; quantification; and sequencing and bioinformatics analysis. The method for constructing a microbial 16S rDNA single-molecule level sequencing library is low in cost and simple in operation, and can simplify experimental processes and reduce sequencing cost.

Description

Method for constructing microbial 16S rDNA single molecule horizontal sequencing library Technical field

The invention relates to the field of microbial gene sequencing, in particular to a method for constructing an amplification primer and a sequencing library for sequencing 16S rDNA single molecule level of bacteria and archaea.

Background technique

16S rDNA is a DNA sequence encoding 16S rRNA and is present in all bacterial genomes. The 16S rDNA sequence consists of nine hypervariable regions (V1-V9) interspersed with conserved regions. Conserved regions have no significant differences between bacteria and can be used to construct a unified evolutionary tree of all life. The hypervariable region has some differences in different bacteria. The 16S rDNA hypervariable region is sequenced, and the microbial identification can be refined to the taxonomic and even species level. By detecting the sequence variation and abundance of 16S rDNA, it can reflect the classification and abundance of bacteria in environmental samples, which has important guiding significance for studying the microbial composition in the environment of marine, soil and intestinal feces.

At present, there are three main methods for 16S rDNA sequencing: single V region (V3/V4/V6), double V region (V3-V4 or V4-V5), and three V regions (V1-V3, V5-V7, V7). -V9), or measure the entire 16S rDNA sequence. At present, the V4 region has the highest accuracy in species identification at each level, but the double V region is longer than the single V region sequence, and the V3-V4 region is the longest of all variable regions, and the combined length is 459 bp, including The amount of information is the largest, the binding specificity of the primers in the V3-V4 region is good, and the coverage in bacteria and archaea is high. The single-sequence can simultaneously detect the diversity distribution of bacteria and archaea, and the double-V region for bacterial classification has been accurate. At the genus level, there is no need to measure the entire 16S rDNA gene. Therefore, the 16S rDNAV3-V4 region sequencing is the best choice for microbial species identification.

Based on Illumina Miseq's 16S rDNA sequencing or Illumina Hiseq platform's metagenomic sequencing, parallel sequencing of multiple samples can be performed in one go, providing environmental sample species classification, species abundance, population structure, phylogenetic evolution, community comparison and much more. At present, the method of sequencing and building the library is mainly based on the two-step method and the one-step method. The application of the one-step method is now more and more, the advantages include simple operation, and can reduce the error and influence caused by operation, and from the perspective of cost. One step is more cost effective. However, the existing one-step amplification does not take into account the effect of the amplification itself on the results, because the preference problem of amplification leads to poor uniformity and cannot accurately reflect the abundance of the objective flora. The advantage of single-molecule 16S rDNA sequencing detection is that it can be accurate to a single metagenomic template, and absolute quantitative abundance analysis can be performed.

The existing single-molecule 16S rDNA sequencing technology mainly uses the sequencing platform of ion torrent. The sequencing principle of ion torrent is to amplify a single template by micro-oil droplets, so it can achieve single molecule level. However, the currently more widely used illumina sequencing platform cannot achieve 16S rDNA single-molecule sequencing analysis.

Therefore, on the basis of maintaining the advantages of the one-step method, it is necessary to develop a library-based sequencing method based on the illumina platform that can be accurate to the 16S rDNA single molecule level.

Summary of invention

The technical problem to be solved by the present invention is to provide a method for constructing a microbial 16S rDNA single molecule horizontal sequencing library with low cost and simple operation.

In a first aspect, the invention provides two primer pairs for library construction, wherein the first primer pair comprises a long primer for use in the first step amplification, including an upstream primer and a downstream primer. The upstream primers include a linker sequence (complementary to the sequence on the sequencing chip), an index sequence (used to distinguish different samples), and a UID random tag (for distinguishing whether the DNA molecule is amplified or not) from the 5' end to the 3' end, respectively. The resulting primer sequences are 10 bp in length, the combination of which is random, and the V3-V4 region is amplified. The downstream primers include a linker sequence (complementary to the sequence on the sequencing chip), an index sequence (optional), and a UID random tag (for distinguishing whether the DNA molecule is amplified or not, and the length is from 5' to 3', respectively. The 10 bp base, the combination of which is random), the V3-V4 region amplification primer sequence. When performing single-ended index sequencing, the downstream primers may be free of index sequences. If double-ended index sequencing is required, the downstream primer can contain an index sequence.

In a specific embodiment, the upstream primer of the first primer pair is any one of the following Tables REP1/3-9, and the downstream primer is any one of the following Tables REP2/10-17.

A second primer pair for library construction contains P1 and P2 short primers. In a specific embodiment, the sequences of the upstream primer P1 and the downstream primer P2 are set forth in SEQ ID NO. 3 and SEQ ID NO. 4, respectively.

In a second aspect, the invention provides a method of constructing a 16S rDNA hypervariable region sequencing library of a microorganism, the method comprising: amplifying a microbial bacterial genome 16S rDNA using the primer pair of the invention to construct a sequencing library.

Further, the method of constructing a 16S rDNA sequencing library of microbial bacteria comprises the following steps:

a) collecting a biological sample and extracting microbial metagenomic DNA in the sample;

b) amplifying the 16S rDNA v3-v4 hypervariable region by PCR using the primer pair of the present invention;

c) purifying the product obtained after amplification to obtain a purified sequencing library;

d) performing accurate quantification and fragment size analysis of the purified sequencing library and sequencing;

e) Bioinformatics analysis of the sequencing results, complete the construction of the microbial 16S RDNA single molecule level sequencing library.

In a specific embodiment, the sample in step a) is a stool sample. Preferably, the concentration and purity are measured using a micro-ultraviolet spectrophotometer after extraction of the sample metagenomic DNA.

Further, the amplification in step b) is a two-step amplification, and the first step: using the first primer pair of the present invention to amplify two cycles using the sample metagenomic DNA as a template to ensure that the two segments are connected with a linker. A complete and single library fragment was generated; the second step amplification was performed using the first amplified product as a template, and the second primer pair of the present invention was used to amplify for 30 cycles to amplify the signal. Preferably, the product obtained after two-step amplification is subjected to agarose gel electrophoresis.

In a specific embodiment, purification is carried out in step c) using AMPure XP magnetic beads from Beckman & Coulter. In a specific embodiment, the purified sequencing library is subjected to accurate quantification and fragment size analysis using Qubit 3.0 and Agilent 2100, and the sequencing library is diluted according to the sequencing platform requirements, and the results of Qubit 3.0 quantification are used as standards and mixed according to the amount of data required. Finally, sequencing was performed using Illumina HiseqX or Miseq.

The method for constructing the 16S rDNA single molecule level sequencing library of the microorganism of the present invention is different from the prior art in that:

1. The primer designed by the invention is a long primer containing a UID random tag, and the two-step amplification can ensure that one label corresponds to the unique amplified 16S rDNA template, and finally the single molecule template can be accurately determined in the analysis of the sequencing result. The level of the 16S rDNA flora abundance and composition can be calculated more accurately.

2. The unique two-step amplification method of the present invention, the 16S rDNA template of the sample to be tested is subjected to the first step of amplification, and the two ends are labeled with a unique label, and the second step amplifies the effective amplification signal but No increase in the number of tags for subsequent sequencing and analysis.

3. The invention realizes the sequencing and analysis of the single molecule 16S rDNA through the illumina sequencing platform, breaks the original situation that the single molecule 16S rDNA can only be analyzed through the ion torrent sequencing platform, simplifies the experimental process and reduces the sequencing cost.

4. The target fragment amplified by the long primer provided by the invention is the V3-V4 region of 16S rDNA, and the amplified fragment is longer in length than the single segment of 16S rDNA only, and contains more information and distinguishes. Higher rate, easy to distinguish between species

DRAWINGS

1 is a schematic diagram of sequences REP1 and REP2 of primers in a first amplification in a method for constructing a 16S rDNA single-molecule level sequencing library of the present invention, wherein N is an uncertain base and N is selected from A, C, G, and T Any one of them; index: split tag sequence; UID: random tag; 16S rDNA primer: 16S rDNA primer sequence;

2 is an agarose gel electrophoresis pattern of a two-step amplification method using a 16S rDNA single-molecule level sequencing library of the microorganism of the present invention, wherein the left side is a product electrophoresis pattern after two-step amplification, and the DNA marker is on the right side. ;

Figure 3 is a 2100 quality control peak map of a 16S rDNA single molecule level sequencing library after magnetic bead purification;

Figure 4 is a graph showing the composition and proportion of the 16S rDNA genus of the gut microbes in the test sample.

Figure 5 shows the composition and proportion of the microbial 16S rDNA genus at the level of the female genital tract secretion and the male faecal sample.

Detailed description of the invention

Embodiments of the present invention, as well as its various features and advantageous details, are described more fully by way of non-limiting embodiments and examples of the embodiments illustrated in the accompanying drawings. It should be noted that the features described in the drawings are not necessarily to scale, and the features of one embodiment can be used with other embodiments, although not explicitly illustrated herein.

definition

Unless otherwise stated, the terms used in the claims and the specification are as defined below.

The term "nucleic acid" refers to a polymer of nucleotides, including any form of DNA or RNA, including, for example, genomic DNA; complementary DNA (cDNA), a DNA representation of mRNA, usually reversed by messenger RNA (mRNA). Recorded, or obtained by amplification; synthetically produced or amplified by production of DNA molecules; and mRNA.

The term "oligonucleotide" refers to a nucleic acid that is relatively short, generally shorter than 200 nucleotides, more specifically shorter than 100 nucleotides, and most particularly shorter than 50 nucleosides. acid. Typically, an oligonucleotide is a single stranded DNA molecule.

The term "primer" refers to an oligonucleotide under suitable conditions (ie, in the presence of four different nucleoside triphosphates and a polymerization reagent (eg, DNA or RNA polymerase or reverse transcriptase) The oligonucleotide is capable of hybridizing to a nucleic acid (also referred to as "annealing") in a suitable buffer and at a suitable temperature and is used as a starting point for nucleotide (RNA or DNA) polymerization. The appropriate length of the primer depends on the intended use of the primer, but typically the primer is at least 7 nucleotides in length, more typically ranging from 10 nucleotides to 30 nucleotides, or even more typically from 15 Nucleotides up to 30 nucleotides in length. Other primers may be slightly longer, for example 30 to 50 nucleotides in length. In this context, "primer length" refers to a portion of an oligonucleotide or nucleic acid that hybridizes to a complementary "target" sequence and initiates nucleotide synthesis. Short primer molecules generally require cooler temperatures to form a sufficiently stable hybrid complex with the template. Primers do not have to reflect the exact sequence of the template but must be sufficiently complementary to hybridize to the template.

The term "primer pair" refers to a set of primers comprising a 5' "upstream primer" or "forward primer" that hybridizes to the complementary sequence at the 5' end of the DNA sequence to be amplified, and to be amplified. A 3' "downstream primer" or "reverse primer" hybridized at the 3' end of the sequence. As will be appreciated by one of ordinary skill in the art, the terms "upstream" and "downstream" or "forward" and "reverse" are not intended to be limiting, but rather provide a schematic orientation.

"Reagent" broadly refers to any reagent used in the reaction other than an analyte, such as a nucleic acid being analyzed. Illustrative reagents for nucleic acid amplification reactions include, but are not limited to, buffers, metal ions, polymerases, reverse transcriptases, primers, template nucleic acids, nucleotides, labels, dyes, nucleases, and the like. Reagents for the enzymatic reaction include, for example, substrates, cofactors, buffers, metal ions, inhibitors, and activators.

The invention is described below based on the examples, but the invention is not limited to only these examples.

Example 1

The method for constructing the 16S rDNA single molecule level sequencing library of the microorganism of the present embodiment is carried out as follows:

(1) Collect normal human feces samples, store them in 1mL fecal preservation solution (can be stored at room temperature for 15 days), and use Tianmu's fecal microbial DNA extraction kit (ZYMO's Quick-DNA ^TM Fecal Microbe Microprep Kit) , according to the steps of the manual to extract fecal macrogenomic DNA;

(2) The extracted metagenomic DNA is detected by a micro-ultraviolet spectrophotometer to determine the concentration and purity of the sample, and the micro-ultraviolet spectrophotometer model is Nanodrop 2000;

(3) The first step of amplification was carried out using Invitrogen's multiplex amplification reagent (Invitrogen's Multiplex PCR master mix); the first step of amplification of 23 μL PCR reaction system is shown in Table 1, the first step of the amplification reaction procedure as As shown in Table 2, the primer sequences are shown in SEQ ID NO. 1 and SEQ ID NO. 2; as shown in FIG. 1;

The first step of amplification uses the fecal microbial DNA as a template to amplify two cycles, ensuring that the two segments are ligated with a complete linker and a single library fragment is produced;

(4) The second step of amplification is to use the first amplified product as a template, and add P1 and P2 short primers at both ends to amplify 30 cycles to amplify the signal. The 50 μL PCR reaction system amplified in the second step is shown in Table 3. The second step of the amplification reaction procedure is shown in Table 4; the sequences of the short primers of P1 and P2 are shown in SEQ ID NO. 3 and SEQ ID NO.

(5) The product obtained after 10uL amplification in two steps was detected by agarose gel electrophoresis; the detection results are shown in Fig. 2. The V3-V4 region has a fragment size of 455 bp, plus a linker and tag sequence at both ends, which is 641 bp in total. The electropherogram shows that the size of the amplified band of the sample is as expected, and the band is bright without a non-specific amplification band.

(6) The product obtained after two-step amplification was purified using Beckman & Coulter's AMPure XP magnetic beads:

Add 75uL magnetic bead suspension to the reaction system, mix thoroughly by pipetting and let stand for 5min at room temperature; take the supernatant and discard it, wash it twice with 80% ethanol, leave it at room temperature for 5 minutes, dry the magnetic beads, and add 20ul of low TE to elute the magnetic The purified sequencing library is obtained from the beads;

(7) The purified sequencing library was accurately quantified and fragment size analyzed by Qubit 3.0 and Agilent 2100. The results are shown in Fig. 3. The sharp peaks of the main peaks are correct in size and no other fragments are contaminated;

(8) Dilute the sequencing library according to the sequencing platform requirements, quantify the results of Qubit 3.0 as the standard and mix the samples according to the data volume, and finally use Illumina HiseqX for sequencing;

(9) Bioinformatics analysis of the sequencing results, that is, the construction of the microbial 16S rDNA single-molecule level sequencing library was completed, and all the duplicated and repeated sequences of the reads were combined, and the single-ended tags were not repeated after the combination.

Table 1 23 μL PCR reaction system amplified in the first step

Composition	Volume/DNA volume
Multiplex PCR master mix (Multiplex Amplification Reagent)	11.5ul
Upstream primer REP1 (10uM)	1ul

Downstream primer REP2 (10uM)	1ul
Microbial genomic DNA	3ng
water	Make up 23ul

Table 2 First step amplification reaction procedure

Table 3 50μL PCR reaction system amplified in the second step

Composition	Volume/DNA volume
First step amplification system	23uL
Multiplex PCR master mix	25uL
Upstream primer P1 at a concentration of 10uM	1uL
Downstream primer P2 with a concentration of 10uM	1uL

Table 4 second step amplification reaction procedure

Example 2

The method for constructing the microbial 16S rDNA single-molecule level sequencing library of the present embodiment is different from that of the first embodiment in that the step (8) is performed by using Miseq, and the step (9) continues to be carried out in the same manner as in the first embodiment. Informatics analysis, the completion of the construction of a microbial 16S rDNA single molecule level sequencing library.

Verification test

A normal human sample was collected, and the experiment was carried out by the procedure described in Example 1. The sequencing results were obtained by sequencing on the Hiseq Xten platform. The GC was between 51-53%, and the number of R1 and R2 reads was about 3 million. The tag is de-duplicated to 600,000 and 900,000 respectively, and the tag repetition rate of R1 is slightly higher than R2. The bidirectional 16S sequence database has a higher ratio of more than 90%. Because it is the Heseq platform sequencing, the length of the clean reads is about 120bp. If you use the Miseq platform, you can test it, bidirectional 300bp, and then splicing. There are more than 600 clusters in the genus after clustering. The specific data is shown in Table 5.

Table 5 Verification test analysis results

It can be seen from the data in Fig. 4 that the proportion of Prevotella is 37% of the total, followed by Blautia (6.7%), Eubacterium (6.5%) and Faecalibacterium (4.0%). Clostridium (3.2%), Copulococcus (2.6%), Bacteroides (2.4%), Ruminococcus (2.1%), and the remaining bacteria are not listed because they are less than 2%. However, the total proportion of the remaining bacteria can account for 34.6%, as shown in Figure 4.

Compared with related literatures or examination reports, the content of Prevotella in the intestine was lower in this study, and the proportion of some rare bacteria (eg. Bifidobacterium) increased, which may be due to conventional amplification. The method further magnifies the proportion of the main flora due to the multiple rounds of amplification preference, while the rare flora has a low proportion, the competitive disadvantage is not effectively amplified, but the proportion is getting lower and lower, so the single molecule amplification method can react more objectively. The actual situation of the microbial composition in the actual intestine.

Example 3

In this example, one normal male stool sample (different from the stool sample collection time in the verification test) and one normal female reproductive tract secretion sample were collected, and the conventional 16S rDNA one-step amplification method was used to construct the library and the method described in this paper. Microbial 16S rDNA single-molecule two-step amplification library construction method is compared according to the following steps:

(1) Fecal samples, stored in 1mL of fecal preservation solution (can be stored at room temperature for 15 days), using Tianmu's fecal microbial DNA extraction kit (ZYMO Quick-DNA ^TM Fecal Microbe Microprep Kit), according to the instructions The steps of extracting fecal macrogenomic DNA; genital secretion samples, stored in 1mL preservation solution and mixed (can be stored at room temperature for 15 days), using Kaijie's DNA extraction kit (Qiagen's QIAamp DNA Mini Kit), according to The steps of the instructions extract total DNA from the genital tract secretions.

(2) The extracted DNA is used to detect the concentration and purity of the sample using a micro-ultraviolet spectrophotometer, and the model of the micro-ultraviolet spectrophotometer is Nanodrop 2000;

(3) One-step and single-molecule two-step amplification were performed using Invitrogen's multiplex amplification reagent (Invitrogen's Multiplex PCR master mix); the two-step amplification procedure is shown in Example 2.

(4) One-step amplification directly amplified 30 cycles using REP1 and REP2.

(5) The product obtained after amplification was purified by AMPure XP magnetic beads of Beckman & Coulter.

(6) The purified sequencing library was accurately quantified and fragment size analyzed using Qubit 3.0 and Agilent 2100;

(7) Dilute the sequencing library according to the sequencing platform requirements, quantify the results of Qubit 3.0 and mix the samples according to the data volume, and finally use Illumina HiseqX for sequencing;

(8) Bioinformatics analysis was performed on the sequencing results, and all the reads with repeated tags and repeated sequences at both ends were combined. The single-ended tags were not duplicated after the combination, and the one-step amplification and sequencing results were not tagged.

From the results shown in Figure 5, the microbial composition of the female reproductive tract is relatively simple, and the lactic acid bacteria account for 99%. The composition and proportion of intestinal microflora are diversified. It can be seen from the figure that the actual proportion of some microflora is exaggerated, such as faecalibacterium, phascolarctobacterium, which actually accounts for only 1%, and the conventional one-step amplification has a preference. Zoom in to 17%. The same one-step amplification of 4% of Blautia is underestimated, in fact, 11%. Therefore, it can be concluded that the single-molecule two-step method is more accurate for the evaluation of the proportion of microbial populations than the traditional one-step method.

While the invention has been described with respect to the preferred embodiments of the present invention, it is understood that the scope of the invention is defined by the appended claims. A person skilled in the art can make various changes or modifications to the embodiments without departing from the spirit and scope of the invention, and these modifications and modifications fall within the scope of the invention.

Claims (9)

1. A primer pair for constructing a microbial 16S rDNA single molecule level sequencing library, comprising a first primer pair consisting of an upstream primer and a downstream primer, wherein the upstream primer comprises from the 5' end to the 3' end, respectively: on the sequencing chip a sequence complementary to the linker sequence, an index sequence for distinguishing different samples, a UID random tag, a V3-V4 region amplification primer sequence, and the downstream primer includes a sequence from the 5' end to the 3' end, respectively: Complementary linker sequences, index sequences for differentiating different samples, UID random tags, V3-V4 region amplification primer sequences.
2. The primer pair according to claim 1, wherein the upstream primer is any one of REP1/3-9, and the downstream primer is any one of REP2/10-17.
3. The primer pair according to claim 1 or 2, further comprising a second primer pair, the upstream primer P1 and the downstream primer shown by SEQ ID NO. 3 and SEQ ID NO. 4, respectively. P2 composition.
4. A method for constructing a microbial 16S rDNA single-molecule horizontal sequencing library, characterized in that a primer pair is used to amplify a microbial bacterial genome 16S rDNA to construct a sequencing library.
5. The method of claim 4 comprising the steps of:

   a) collecting a biological sample and extracting microbial metagenomic DNA in the sample;

   b) amplifying the 16S rDNA v3-v4 hypervariable region by PCR using the primer set of claim 3;

   c) purifying the product obtained after amplification to obtain a purified sequencing library;

   d) performing accurate quantification and fragment size analysis of the purified sequencing library and sequencing;

   e) Bioinformatics analysis of the sequencing results, complete the construction of the microbial 16S RDNA single molecule level sequencing library.
6. The method according to claim 5, wherein the sample in step a) is a stool sample, preferably, after extracting the sample metagenomic DNA, its concentration and purity are detected using a micro-ultraviolet spectrophotometer.
7. The method according to claim 5, wherein said amplification in step b) is a two-step amplification, and the first step: using the first primer pair according to claim 1 or 2 to amplify the sample metagenomic DNA as a template Two cycles, ensuring that the two segments are ligated with a complete and a single library fragment; the second step amplifies the first amplified product as a template and the second primer pair of claim 3 for 30 cycles. To amplify the signal; preferably, the product obtained after two-step amplification is subjected to agarose gel electrophoresis.
8. The method according to claim 5, wherein the purification is carried out in step c) with AMPure XP magnetic beads from Beckman & Coulter, preferably, the purified sequencing library is subjected to accurate quantification and fragment size analysis using Qubit 3.0 and Agilent 2100, according to sequencing. The platform required dilution of the sequencing library, quantification of the results of Qubit 3.0 and mixing according to the amount of data required, and finally sequencing with Illumina HiseqX or Miseq.
9. The method of claim 5 comprising the steps of:

   (1) Collecting normal human feces samples, storing them in 1 mL of fecal preservation solution, using Tianmu's fecal microbial DNA extraction kit, and extracting fecal macrogenomic DNA according to the instructions;

   (2) The extracted metagenomic DNA is detected using a micro-ultraviolet spectrophotometer to determine the concentration and purity of the sample;

   (3) using the multiplex amplification reagent of Yingjie Company for the first step amplification;

   The first step of amplification uses the fecal microbial DNA as a template to amplify two cycles, ensuring that the two segments are ligated with a complete and single library fragment; the upstream primer REP1 and the downstream primer REP2 sequences are SEQ ID NO. 1 and SEQ ID NO, respectively. .2;

   (4) The second step of amplification uses the first amplified product as a template, and adds P1 and P2 short primers at both ends to amplify 30 cycles to amplify the signal; the sequences of the upstream primer P1 and the downstream primer P2 are respectively SEQ ID NO. .3 and SEQ ID NO. 4;

   (5) taking 10uL after two-step amplification, the product was detected by agarose gel electrophoresis;

   (6) The product obtained after two-step amplification was purified using Beckman & Coulter's AMPure XP magnetic beads:

   Add 75uL magnetic bead suspension to the reaction system, mix thoroughly by pipetting and let stand for 5min at room temperature; take the supernatant and discard it, wash it twice with 80% ethanol, leave it at room temperature for 5 minutes, dry the magnetic beads, and add 20ul of low TE to elute the magnetic The purified sequencing library is obtained from the beads;

   (7) The purified sequencing library was accurately quantified and fragment size analyzed using Qubit 3.0 and Agilent 2100;

   (8) Dilute the sequencing library according to the sequencing platform requirements, quantify the results of Qubit 3.0 and mix the samples according to the data volume, and finally use Illumina HiseqX or Miseq for sequencing;

   (9) Bioinformatics analysis of the sequencing results, that is, the construction of the microbial 16S RDNA single-molecule level sequencing library was completed, and all the duplicated and repeated sequences of the reads were combined, and the single-ended tags were not repeated after the combination.

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