CN113030548A - Method and device for extracting characteristic information of nanopore detection current signal based on local threshold - Google Patents
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Abstract
The invention discloses a method and a device for extracting characteristic information of a nanopore detection current signal based on a local threshold, which are used for acquiring ion current signal data of a nanopore sensor in a period of time in advance, selecting the local data in a moving window, and setting a baseline and a double threshold so as to preliminarily judge a starting point and an end point of a pulse signal; fitting the preliminarily selected pulse electrical signal data by utilizing Fourier series, correcting by a second-order difference and rise time method, and further screening pulse signals caused by molecular translocation in the nano holes; extracting characteristic information of the pulse signal caused by the judged molecular translocation event; and selecting multi-dimensional parameter information extracted from the molecular characteristic events of the signals, storing and outputting the information, performing statistical analysis, and processing nanopore electric signal data with high flux. The invention has high precision and batch processing of data, realizes high-throughput automatic analysis of the big data of the nanopore sensor, and is beneficial to the wide application of the nanopore sensor.
Description
Technical Field
The invention belongs to the field of signal processing and software design, and particularly relates to a nanopore detection current signal characteristic information extraction method and device based on a local threshold in a mobile window hole.
Background
The nanopore sensor is used as a single molecule detection tool and is more and more widely applied to the field of biosensing detection analysis, has the advantages of no need of marking and amplification, low cost, sensitivity, high flux and the like, and can be used for detecting various unlabelled biomolecules, such as DNA, RNA, protein, ligand or protein-DNA compound and the like. Nanopore detection is simple, and when molecules pass through the nanopore, ion current is increased or decreased, a series of pulse signals are generated, and information such as the size, charge and conformation of an analyte is hidden in the molecular event pulse signals. Therefore, the judgment and information extraction of a large number of current pulse signals obtained by a nanopore sensing experiment are very important. The existing information extraction processing method for detecting a current signal by a nanopore is limited, for example, a threshold setting and template method is mainly adopted in the calmfit, a threshold or template event is selected, and a pulse signal caused by a molecule translocation event is determined simply according to a difference or comparison. The method has certain recognition degree on long-time rectangular signals caused by linear molecule through holes with the same length as DNA. However, when the baseline current noise is large, especially the molecular via speed is fast, and the pulse signal time is short, the pulse signal caused by the molecular translocation event is often difficult to identify, and even many false signals occur, and the reading and output of the information are limited.
At present, the detection range of the nanopore sensor is expanded, signal changes caused by translocation of different molecules are more various, and the acquired pulse signals are more complex, so that the development of a novel efficient and accurate information extraction method for nanopore current detection signals is particularly urgent. On the basis, the invention designs a self-adaptive molecular characteristic event judgment and extraction method of the sensor electric signal based on the matlab tool, and through setting a signal window and a double-threshold judgment standard, the selected data segment is subjected to pulse signal identification, and meanwhile, the data is fitted and corrected, so that signal interference such as background noise is eliminated. Once the pulse signal of the nanopore sensor is determined, the method further extracts, stores and outputs the parameters such as the retention time, the signal pulse amplitude and the signal integral area of the whole event of the characteristic event, and provides functions such as a statistical analysis graph of each parameter of the characteristic event.
Disclosure of Invention
The purpose of the invention is as follows: the invention provides a method and a device for extracting characteristic information of a nanopore detection current signal based on a local threshold, which mainly extract the characteristic information of a current pulse signal detected by a nanopore sensor and realize the functions of characteristic information acquisition, signal analysis characterization and the like of the detected translocation molecule pulse signal.
The invention content is as follows: the invention provides a nanopore detection current signal characteristic information extraction method based on a local threshold, which specifically comprises the following steps:
(1) acquiring ion current signal data of the nanopore sensor in advance within a period of time, selecting local area data in a moving window, and setting a baseline and a double threshold value, so as to preliminarily determine a starting point and an ending point of a pulse signal, wherein each pulse signal represents a blocking current change caused by the passage of a detection molecule in a nanopore and is called a molecule translocation event;
(2) fitting the preliminarily selected pulse electrical signal data by utilizing Fourier series, correcting by a second-order difference and rise time method, and further screening pulse signals caused by molecular translocation in the nano holes;
(3) when the pulse signal is judged as the nanopore molecule characteristic event, extracting characteristic information of the pulse signal caused by the judged molecule translocation event, wherein the characteristic information comprises information such as retention time, amplitude, peak position, voltage, signal integral and the like of the pulse signal;
(4) and storing and outputting the multidimensional parameter information extracted from the molecular characteristic events of the selected signals, performing statistical analysis, and processing nanopore electric signal data with high flux.
Further, the step (1) is realized as follows:
the nanopore ionic current is:
wherein, I0(t) is the baseline current (I),
is an event current, In(t) is current noise; in order to minimize the influence of baseline drift, the loaded electric signal data is read in a segmented mode, baseline setting is carried out on the data in a local area, and the window width (ww) is set; setting data points in a window as i, setting baseline current as the mean value of current values of all data points in the window, estimating local baseline according to moving window average, and setting the local baseline as baseline (i); detecting that the initial state is 0, and judging from the first point after the window (i ═ ww + 1); setting a minimum threshold u according to a change in a pulse signal0And maximum threshold u1Determining the molecular event, local threshold ujBy subtracting a constant threshold u from the local baseline0To define:
wherein, IkIs the current value at data point k, window width ω; when the molecule access hole is blocked, the ion current in the hole is reduced, so the current value of the event starting point in the window is smaller than the baseline current, and the difference value between the current value and the baseline current is larger than the initially set lower threshold value and smaller than the upper threshold value (u)0<Ik<u1) Data points (data (i,2)<baseline(i))&&(data(i-2,2)>baseline (i-2)); when the current value of the data point detected in the window returns to the baseline current (data (i,2)>baseline (i)), the event ends.
Further, the step (2) comprises the steps of:
(21) a smoothing method based on fitting experimental blocking signals to a fourier series:
wherein, aj，βjAnd ω is the coefficient of the Fourier series, n is the order of the model; the Fourier series decomposes the blocking signal into the sum of a group of simple oscillating functions;
(22) performing two differential operations on the event data after fitting, solving extreme points of newly obtained data, correcting the initial point from the beginning to the end of the event, setting the previous extreme point as a new event initial point when a current value between two extreme points meets a condition of being smaller than the current mean value of the whole fitting event, and performing operations from the beginning to the end of the fitting event when subsequent extreme points are not considered any more; correcting the termination point just in the opposite way, and setting a first extreme point between the current value and the baseline current difference between the upper threshold value and the lower threshold value as a new termination point from the end of the event to the beginning of the event;
(23) the rise time Tr of the low pass filter prevents the blocking signal from reaching its true minimum or plateau:
Tr＝0.3321/fc
wherein f iscIs the frequency of the filter, and the low-pass filter can eliminate short-term fluctuation such as the shape deformation of the blocking signal.
Further, the step (3) is realized as follows:
td＝te-ts
Ipeak＝|Ibase-Imin|
wherein, tdFor pulse signal retention time, t is determined by determining the start and end points of molecular eventss、teRespectively a start point and a stop point of the blocking signal; i ispeakIs a peak value, Ibase、IminData baseline current and minimum within an event, respectively; amplitude IcIs based on the average of data points within the occlusion signal in the molecular event
And IpeakApproaching; i isctdIntegration of the signal for each event, IkIs the current value for data point k.
Based on the same inventive concept, the invention also provides a nanopore detection current signal feature extraction device based on the local threshold, which comprises a memory, a processor and a computer program, wherein the computer program is stored on the memory and can run on the processor; the computer program, when loaded into a processor, implements the method for nanopore detection current signal feature extraction based on local threshold.
Has the advantages that: compared with the prior art, the invention has the beneficial effects that: 1. the current signals are processed in batches by a window moving method, the current of a base line in the area is relatively stable, and the efficiency and the accuracy of processing the signals are effectively improved; 2. the upper threshold and the lower threshold are adopted to judge the signal event, and a second-order difference method and a rise time correction method are adopted simultaneously, so that the accuracy of information extraction is improved; 3. the obtained pulse signal information caused by molecular translocation is richer, and the information such as duration, amplitude, current integral value, voltage and the like is provided, so that the establishment of a molecular event classification database is facilitated; 4. by means of a self-contained program of the matlab program, the method has self-adaptability, flexibly sets parameters and facilitates multi-dimensional statistical analysis.
Drawings
FIG. 1 is a schematic diagram of a nanopore sensor sensing a current pulse signal;
FIG. 2 is a graph of pulse signals resulting from a molecular translocation event;
FIG. 3 is a schematic diagram illustrating a time starting point determination;
FIG. 4 is a diagram illustrating a calibration of a rising time starting point;
FIG. 5 is a diagram illustrating the correction of the starting point of the second-order difference DBC;
FIG. 6 is a diagram of a nanopore pulsed signal molecular event recognition method interface;
FIG. 7 is a nanopore pulsed signal molecule event signature information extraction graph, wherein (a) is a list of events; (b) is a single event image; (c) is signal characteristic information;
FIG. 8 is a scatter plot of the distribution of residence time and amplitude of extracted molecular events within an analysis window.
Detailed Description
The technical scheme of the invention is clearly and completely described below with reference to the accompanying drawings.
The invention provides a nanopore detection current signal characteristic information extraction method based on a local threshold, which specifically comprises the following steps as shown in figure 1:
step 1: the ion current signal data of the nanopore sensor in a period of time is obtained in advance, local area data in a moving window is selected, baseline and double threshold setting is carried out, and therefore the starting point and the ending point of a pulse signal are preliminarily determined, as shown in fig. 3, each pulse signal represents the change of blocking current caused by the passage of a detection molecule in the nanopore, and the change is called a molecule translocation event.
The pulse signal caused by the molecular pull-off event is shown in fig. 2, and the nanopore ionic current is:
wherein, I0(t) is the baseline current (I),
is an event current, In(t) is current noise.
To minimize the effects of baseline drift, one willReading the loaded electric signal data in a segmented manner, setting a base line for the data in the local area, and setting the window width (ww); the data points in the window are set as i, the baseline current is the mean value of the current values of all the data points in the window, the local baseline is estimated according to the moving window average, and the baseline current is set as baseline (i). Detecting that the initial state is 0, and judging from the first point after the window (i ═ ww + 1); setting a minimum threshold u according to a change in a pulse signal0And maximum threshold u1Determining the molecular event, local threshold ujBy subtracting a constant threshold u from the local baseline0To define:
wherein, IkIs the current value at data point k, window width ω.
When the molecule access hole is blocked, the ion current in the hole is reduced, so the current value of the event starting point in the window is smaller than the baseline current, and the difference value between the current value and the baseline current is larger than the initially set lower threshold value and smaller than the upper threshold value (u)0<Ik<u1) Data points (data (i,2)<baseline(i))&&(data(i-2,2)>baseline (i-2)); when the current value of the data point detected in the window returns to the baseline current (data (i,2)>baseline (i)), the event ends.
Step 2: fitting the preliminarily selected pulse electrical signal data by utilizing Fourier series, correcting by a second-order difference and rise time method, and further screening pulse signals caused by molecular translocation in the nano-pores.
The nanopore electric signal has a sampling frequency of more than 100kHz, large data volume and certain background noise. To avoid the influence of noise, a smoothing method based on fitting experimental blocking signals to a fourier series is applied:
wherein, aj，βjAnd ω is the coefficient of the Fourier series, and n is the order of the model. The fourier series decomposes the blocking signal into a sum of a set of simple oscillating functions (sine and cosine). The minimum of the second order differential can be easily identified by applying a fourier smoothing function.
As shown in fig. 4, based on the fitted event signal data, the extreme value data point is obtained, and two thresholds u are set0And u1The correction of the starting point is from the beginning to the end of the event, when an extreme value satisfies that the difference value between the current value and the baseline current is between the upper threshold value and the lower threshold value, the extreme value is set as a new starting point, the subsequent extreme value point is not considered any more, the correction of the ending point is just opposite, and from the end of the event to the beginning, the first extreme value satisfying the difference value between the current value and the baseline current and the upper threshold value and the lower threshold value is set as a new ending point.
As shown in fig. 5, two differential operations are performed based on the fitted event data, and an extreme point of the newly obtained data is obtained, when a current value between two extreme points satisfies a condition smaller than a current mean of the entire fitted event, the previous extreme point is set as a new event starting point, and the subsequent extreme points are not considered any more, and the operations are performed from the beginning to the end of the fitted event.
The rise time (Tr) of the low pass filter prevents the blocking signal from reaching its true minimum or plateau. In general, blocking signals with residence times less than 2Tr are severely attenuated. Tr of the filter can be estimated by:
Tr＝0.3321/fc
where fc is the frequency of the filter. The low pass filter can eliminate short term fluctuations such as flutter noise, for example, the shape of the blocking signal is distorted. The rise time of the filter will result in a prolonged blocking signal.
And step 3: when the pulse signal is judged as the nanopore molecule characteristic event, characteristic information extraction is carried out on the pulse signal caused by the judged molecule translocation event, wherein the characteristic information comprises information such as retention time, amplitude, peak position, voltage and signal integration of the pulse signal.
Pulse signal dwell time tdDependent primarily on determining molecular eventsA start point and an end point, which will be set as the start (t) of the blocking signals) And stop (t)e) Points, i.e. td＝te-tsThe dwell time is therefore the time interval from the start point to the end point of the event. Peak is the difference between the minimum of the data within the event and the baseline, Ipeak＝|Ibase-IminL. And amplitude IcIs based on the average of data points within the occlusion signal in the molecular event
And IpeakAnd (4) approaching. Signal integration per event
Wherein t isdIs the residence time of the event, IbaseIs a base line current, IkIs the current value for data point k.
And 4, step 4: and storing and outputting the multidimensional parameter information extracted from the molecular characteristic events of the selected signals, performing statistical analysis, and processing nanopore electric signal data with high flux.
Based on the same inventive concept, the invention also provides a nanopore detection current signal feature extraction device based on the local threshold, which comprises a memory, a processor and a computer program, wherein the computer program is stored on the memory and can run on the processor; the computer program, when loaded into a processor, implements the method for nanopore detection current signal feature extraction based on local threshold.
Based on MATLAB GUI (Graphical User Interface) design, the pulse signals of the nanopore detection current are identified and information is extracted and analyzed by adopting threshold setting of local area data in a moving window. The process mainly comprises the steps of threshold setting, data correction, information extraction, result display and the like. The basic principle for judging the molecular via event is to observe the fluctuation amplitude of the changed current signal relative to the baseline current, so the setting of the dual threshold is very important. Through setting a signal window and a double-threshold judgment standard, pulse signals are identified for the selected data segment, and meanwhile, fitting and correcting are carried out on the data, so that signal interference such as background noise is eliminated. Once the current pulse signal of the nanopore sensor is determined, parameters such as the retention time of the characteristic event, the signal pulse amplitude, the signal integral area of the whole event and the like are further extracted, stored and output, and a statistical and analytical map of the molecular event characteristic information is provided.
The main implementation operational interface is shown in fig. 6. And opening a main program, loading data, and displaying the signals in a coordinate system, wherein the horizontal axis of the coordinate system is time, and the vertical axis of the coordinate system is current amplitude. As can be seen from fig. 6, the signal we cut is a segment of the original signal between about 60s and 70 s.
After the data loading is completed, signal feature extraction analysis is performed, and translocation events in the window are found out firstly. The principle is based on a moving window averaging algorithm to obtain a baseline current for each window, locate a single blocking signal according to a set threshold, and list all translocation events. Here the window width of the moving window averaging method we set to 200. We set two thresholds to screen events, one low threshold u0I.e. when the current amplitude exceeds u with respect to the base line current variation0It is preliminarily judged to be a translocation event. And a high threshold u1And the device is used for filtering the abnormal signals with the too high amplitude. After the threshold value is set, the DBC method and the rising time correction are selected in the event correction, so that the detected translocation event can be corrected, and the detection result is more accurate.
The "Start" button is clicked to Start the analysis process. And waiting for a moment, and displaying a prompt tone and a prompt window to show that the processing is finished. All events found are displayed in the event list. In the list shown in fig. 7, each event has a sequence number, and the length is calculated, where the length refers to the number of data points in the event. Clicking any event in the event list can display the image of the event blocking signal. As shown in fig. 7(a) and 7(b), the single blocking signal of the 40 th event is shown, wherein the black line is the baseline current obtained based on the window-moving average algorithm, the black dotted line is the single blocking signal before uncorrecting, and the black solid line is the actual part of the corrected single blocking signal. The signals were also fitted, with black dashed lines in the image. All signal characteristic information is then shown in the results of fig. 7 (c): dwell time, amplitude, current integration value, peak value, bias voltage. The amplitude obtained here is based on the average of all data points in the event, and the peak is the difference between the minimum of the event data points and the baseline, so the peak is obtained to be larger than the amplitude.
Clicking on the "scatter" button on the interface can draw a scatter plot for all events, as shown in FIG. 8. The x-axis is the residence time of the event and the y-axis is the magnitude of the event. As can be seen, the residence time is centered between 0-0.5 microseconds, with the most intense amplitudes between 4-6 pA. It is based on the statistics of the large number of single blocking signals to detect the molecules, and it can be seen that the residence time is in the microsecond level, which indicates that the molecule passing speed is extremely fast, which is also the main difficulty of signal acquisition and identification. In addition, by clicking the 'save' and 'save fixed data' on the operation interface, the signal characteristic information of all events and the data before and after fitting can be stored in a text or table form, so that further analysis is facilitated.
Claims (5)
1. A nanopore detection current signal characteristic information extraction method based on a local threshold is characterized by comprising the following steps:
(1) acquiring ion current signal data of the nanopore sensor in advance within a period of time, selecting local area data in a moving window, and setting a baseline and a double threshold value, so as to preliminarily determine a starting point and an ending point of a pulse signal, wherein each pulse signal represents a blocking current change caused by the passage of a detection molecule in a nanopore and is called a molecule translocation event;
(2) fitting the preliminarily selected pulse electrical signal data by utilizing Fourier series, correcting by a second-order difference and rise time method, and further screening pulse signals caused by molecular translocation in the nano holes;
(3) when the pulse signal is judged as the nanopore molecule characteristic event, extracting characteristic information of the pulse signal caused by the judged molecule translocation event, wherein the characteristic information comprises information such as retention time, amplitude, peak position, voltage, signal integral and the like of the pulse signal;
(4) and storing and outputting the multidimensional parameter information extracted from the molecular characteristic events of the selected signals, performing statistical analysis, and processing nanopore electric signal data with high flux.
2. The method for extracting the characteristic information of the nanopore detection current signal based on the dual threshold value of claim 1, wherein the step (1) is realized by the following steps:
the nanopore ionic current is:
wherein, I0(t) is the baseline current (I),
is an event current, In(t) is current noise; in order to minimize the influence of baseline drift, the loaded electric signal data is read in a segmented mode, baseline setting is carried out on the data in a local area, and the window width (ww) is set; setting data points in a window as i, setting baseline current as the mean value of current values of all data points in the window, estimating local baseline according to moving window average, and setting the local baseline as baseline (i); detecting that the initial state is 0, and judging from the first point after the window (i ═ ww + 1); setting a minimum threshold u according to a change in a pulse signal0And maximum threshold u1Determining the molecular event, local threshold ujBy subtracting a constant threshold u from the local baseline0To define:
wherein, IkIs the current value at data point k, window width ω; when the molecular access hole is blocked, the ion current in the hole decreases, so that the event starting point in the windowShould be smaller than the baseline current, and should have a difference from the baseline current greater than an initially set lower threshold and less than an upper threshold (u)0<Ik<u1) Data points (data (i,2)<baseline(i))&&(data(i-2,2)>baseline (i-2)); when the current value of the data point detected in the window returns to the baseline current (data (i,2)>baseline (i)), the event ends.
3. The dual-threshold-based nanopore detection current signal characteristic information extraction method according to claim 1, wherein the step (2) comprises the steps of:
(21) a smoothing method based on fitting experimental blocking signals to a fourier series:
wherein, aj，βjAnd ω is the coefficient of the Fourier series, n is the order of the model; the Fourier series decomposes the blocking signal into the sum of a group of simple oscillating functions;
(22) performing two differential operations on the event data after fitting, solving extreme points of newly obtained data, correcting the initial point from the beginning to the end of the event, setting the previous extreme point as a new event initial point when a current value between two extreme points meets a condition of being smaller than the current mean value of the whole fitting event, and performing operations from the beginning to the end of the fitting event when subsequent extreme points are not considered any more; correcting the termination point just in the opposite way, and setting a first extreme point between the current value and the baseline current difference between the upper threshold value and the lower threshold value as a new termination point from the end of the event to the beginning of the event;
(23) the rise time Tr of the low pass filter prevents the blocking signal from reaching its true minimum or plateau:
Tr＝0.3321/fc
wherein f iscIs the frequency of the filter, and the low-pass filter can eliminate short-term fluctuation such as the shape deformation of the blocking signal.
4. The method for extracting the characteristic information of the nanopore detection current signal based on the dual threshold value of claim 1, wherein the step (3) is realized by the following steps:
td＝te-ts
Ipeak＝|Ibase-Imin|
wherein, tdFor pulse signal retention time, t is determined by determining the start and end points of molecular eventss、teRespectively a start point and a stop point of the blocking signal; i ispeakIs a peak value, Ibase、IminData baseline current and minimum within an event, respectively; amplitude IcIs based on the average of data points within the occlusion signal in the molecular event
And IpeakApproaching; i isctdIntegration of the signal for each event, IkIs the current value for data point k.
5. A local threshold-based nanopore detection current signal feature extraction device comprising a memory, a processor and a computer program stored on the memory and executable on the processor, wherein the computer program when loaded into the processor implements the local threshold-based nanopore detection current signal feature extraction method according to any of claims 1-4.
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