CN119026064A - A signal processing and determination method based on capillary electrophoresis nucleic acid fragment analysis - Google Patents
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- 238000004458 analytical method Methods 0.000 title claims abstract description 65
- 238000000034 method Methods 0.000 title claims abstract description 27
- 150000007523 nucleic acids Chemical group 0.000 title claims abstract description 25
- 238000005251 capillar electrophoresis Methods 0.000 title claims abstract description 24
- 238000012545 processing Methods 0.000 title claims abstract description 21
- 238000005070 sampling Methods 0.000 claims abstract description 10
- 238000007781 pre-processing Methods 0.000 claims abstract description 7
- 238000003752 polymerase chain reaction Methods 0.000 claims abstract description 5
- 238000005457 optimization Methods 0.000 claims abstract description 4
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- 230000001066 destructive effect Effects 0.000 claims 1
- 230000002452 interceptive effect Effects 0.000 description 7
- 239000012634 fragment Substances 0.000 description 6
- 238000010586 diagram Methods 0.000 description 4
- 238000001514 detection method Methods 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003086 colorant Substances 0.000 description 2
- 238000012937 correction Methods 0.000 description 2
- 238000003205 genotyping method Methods 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
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- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000001215 fluorescent labelling Methods 0.000 description 1
- 238000012252 genetic analysis Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000007403 mPCR Methods 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
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- 102000004169 proteins and genes Human genes 0.000 description 1
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- G06F—ELECTRIC DIGITAL DATA PROCESSING
- G06F2218/00—Aspects of pattern recognition specially adapted for signal processing
- G06F2218/02—Preprocessing
- G06F2218/04—Denoising
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- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06F—ELECTRIC DIGITAL DATA PROCESSING
- G06F2218/00—Aspects of pattern recognition specially adapted for signal processing
- G06F2218/08—Feature extraction
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Abstract
The invention discloses a signal processing and judging method based on capillary electrophoresis nucleic acid fragment analysis, which relates to the technical field of biological information and comprises the following steps: acquiring an original waveform sampling signal after capillary electrophoresis treatment after multiple polymerase chain reaction; preprocessing an original waveform sampling signal to obtain peak type characteristic point search after peak type optimization; performing preliminary screening on the signal peak target points under comparison judgment on the peak type characteristic points based on a preset position interval and a peak height threshold value; and (3) carrying out proportional combination analysis according to the characteristic points obtained by searching, and carrying out target marking under secondary judgment on the signal peaks based on analysis results. According to the method, the specific algorithm is set to carry out secondary identification according to the characteristics of the shape, the height and the like of the signal peak, so that the accuracy of target spot judgment is greatly improved, and a user can select one algorithm or a plurality of algorithms to start according to actual conditions by combining the three algorithms, so that higher flexibility is provided.
Description
Technical Field
The invention relates to the technical field of biological information, in particular to a signal processing and judging method based on capillary electrophoresis nucleic acid fragment analysis.
Background
Fragment analysis (FRAGMENT ANALYSIS, FA) is a molecular biology technique that performs nucleic acid analysis by measuring nucleic acid fragments of different sizes of fluorescent markers, and is widely used in genotyping, DNA fingerprinting, mutation detection, etc. in the fields of medicine, environmental, agricultural research, etc.
The principle is to design primers aiming at different target targets, and the amplified products of each target have at least 1bp difference. Compared with a third party, the primer is synthesized and fluorescence is marked at the tail end, and fragments with different sizes are marked by adopting fluorescence with the same or different colors, so that multiplex Polymerase Chain Reaction (PCR) is carried out, and the nucleic acid fragments of all targets are marked by fluorescence. After pretreatment of the products, capillary electrophoresis is carried out, and fluorescent labeled products with different fragment sizes and different colors are distinguished and identified by using a fluorescence detector.
In order to realize full-automatic analysis, research on nucleic acid fragment analysis and development of corresponding software are started in foreign countries, and currently, relatively well-known commercial software is the software attached to a gene analyzer developed by ABI corporation of America. The software can analyze and display the collected original data, and identify the position and peak height of each peak of the electropherogram, so as to determine the length (peak position) and the corresponding content (peak height) of the nucleic acid fragment. And comparing the obtained peak position and peak height with a preset position interval and a peak height threshold value, thereby completing the identification of the target point.
In the fragment analysis process, because of the problems of nucleic acid concentration, instrument state fluctuation or reagent consumable quality and the like, baseline disturbance occurs, interference peaks appear in a preset position interval, false positives are easy to appear only through single peak height threshold judgment, and therefore great trouble is caused to interpretation of target targets.
Disclosure of Invention
In order to more accurately identify a target spot and avoid misdiscrimination, the invention provides a signal processing and judging method based on capillary electrophoresis nucleic acid fragment analysis, which comprises the following steps:
S1: acquiring an original waveform sampling signal after capillary electrophoresis treatment after multiple polymerase chain reaction;
S2: preprocessing an original waveform sampling signal to obtain peak type characteristic point search after peak type optimization;
S3: performing preliminary screening on the signal peak target points under comparison judgment on the peak type characteristic points based on a preset position interval and a peak height threshold value;
s4: and (3) carrying out proportional combination analysis according to the characteristic points obtained by searching, and carrying out target marking under secondary judgment on the signal peaks based on analysis results.
Further, in the step S2, the preprocessing includes baseline subtraction and peak height lossless filtering.
Further, the baseline subtraction uses a window sliding minimum extraction method to remove abnormal interference baseline signals.
Further, the peak height lossless filtering adopts median filtering and polynomial smoothing to remove the miscellaneous peak.
Further, in the step S2, the peak-type feature point is a first derivative inflection point of the sampled signal.
Further, in the step S4, the ratio combination analysis includes peak height ratio analysis, peak area/peak height ratio analysis, and mirror image ratio analysis.
Further, the peak height ratio analysis is specifically:
And extracting the peak height of a target mark signal peak, comparing the peak height with the peak height in a preset distance range to obtain signal-to-noise ratio data, and if the signal-to-noise ratio data contains a signal-to-noise ratio smaller than a first set threshold value, judging the signal peak as an interference signal and canceling the target mark.
Further, the peak area/peak height ratio analysis is specifically:
and extracting peak height and peak area of a target mark signal peak, and judging the signal peak as an interference signal and canceling target mark if the ratio of the peak area to the peak height is larger than a second set threshold value.
Further, the mirror image proportion analysis specifically includes:
and taking the peak width of the target mark signal peak as an extraction range, taking the peak top position of the target mark signal peak as an extraction starting point, acquiring waveform signals of peaks in the extraction range around the extraction starting point, acquiring a correlation coefficient after mirror image processing, and judging the signal peak as an interference signal and canceling target mark if the correlation coefficient is smaller than a third set threshold value.
Compared with the prior art, the invention at least has the following beneficial effects:
(1) According to the signal processing and judging method based on capillary electrophoresis nucleic acid fragment analysis, a specific algorithm is set to carry out secondary identification according to the characteristics of the shape, the height and the like of a signal peak, so that the accuracy of target spot judgment is greatly improved, experimental staff is assisted in carrying out result judgment, and the working efficiency is remarkably improved;
(2) Three different algorithms are provided for identifying the signal peaks, and a user can select one or more algorithms to start according to actual conditions, so that high flexibility is provided;
(3) The linear compensation of the amplitude of the peak type interval of the filtered signal is used for eliminating the signal loss in the filtering process, so that the accuracy of the signal peak height can be ensured, and the change of the real signal intensity caused by filtering is prevented.
Drawings
FIG. 1 is a step diagram of a signal processing and determination method based on capillary electrophoresis nucleic acid fragment analysis;
FIG. 2 is an original waveform sample signal;
FIG. 3 is a graph showing signals before and after baseline subtraction correction;
FIG. 4 is a schematic diagram of the signal before and after peak height lossless filtering;
FIG. 5 is a peak diagram of peak feature point search peak points and labeling;
FIG. 6 is a graph showing the result of recognition before and after the start of peak height ratio analysis;
FIG. 7 is a plot of the s/h ratio of normal and interfering peaks;
FIG. 8 is a graph showing the result of recognition before and after the start of the peak area/peak height ratio analysis;
FIG. 9 is a graph of the recognition results before and after opening of the mirror image ratio analysis;
Description of the drawings: in the drawing, the abscissa of the coordinate system is the size of a segment, and the ordinate is the peak height.
Detailed Description
The following are specific embodiments of the present invention and the technical solutions of the present invention will be further described with reference to the accompanying drawings, but the present invention is not limited to these embodiments.
In bioscience research, capillary electrophoresis is often used to separate and analyze biological macromolecules such as proteins, amino acids, polypeptides, nucleotides, and the like. In particular, in the aspect of genetic analysis, it can be used for genotyping, DNA fingerprint analysis, mutation detection and the like. By combining fluorescent labeling techniques, the detection sensitivity can be enhanced, allowing specific sequences in a microsample to be clearly resolved. In order to solve the problem of target spot interpretation interference caused by the problems of nucleic acid concentration, instrument state fluctuation and the like in the fragment analysis process, as shown in fig. 1, the invention provides a signal processing and judging method based on capillary electrophoresis nucleic acid fragment analysis, which comprises the following steps:
S1: acquiring an original waveform sampling signal after capillary electrophoresis treatment after multiple polymerase chain reaction;
S2: preprocessing an original waveform sampling signal to obtain peak type characteristic point search after peak type optimization;
S3: performing preliminary screening on the signal peak target points under comparison judgment on the peak type characteristic points based on a preset position interval and a peak height threshold value;
s4: and (3) carrying out proportional combination analysis according to the characteristic points obtained by searching, and carrying out target marking under secondary judgment on the signal peaks based on analysis results.
In the invention, after the original waveform sampling signal (shown in fig. 2) is obtained, the difference of signal baselines between different channels of the original signal is considered, so that the consistency of channel space signal values is required to be ensured through baseline subtraction. For this reason, we use the method of window sliding minimum extraction, and the window width generally ensures that there are necessarily baseline signal points in the window in most cases. Thus, the window sliding step length is 1 half-peak width, taking a length of 10 consecutive half-peak widths. And outputting a window minimum value by sliding one window, and finally obtaining a group of window minimum value sequences by continuous sliding, and performing median filtering and low-pass filtering on the sequences to remove the baseline signals of abnormal interference. As shown in fig. 3, the signals before and after baseline subtraction correction are shown.
Then, we perform median filtering (filter window width is set) +sg filtering (polynomial smoothing process, polynomial order is set, filter window width is set) on the signal after the baseline subtraction, and eliminate the miscellaneous peak caused by the baseline jump by filtering to smooth the signal. The filtered peak signal is gain amplified to ensure that the peak height value is unchanged. As shown in fig. 4, a signal diagram before and after peak height lossless filtering is shown.
After preprocessing the signal, we need to search the characteristic point of the peak to facilitate the identification of the subsequent interference signal. Specifically, as shown in fig. 5, peak position searching is performed on the signal above the dynamic threshold according to the signal after lossless filtering, the zero crossing point of the corresponding first derivative is performed, and the first derivative around the zero crossing point is monotonically decreased. The peak foot point is preliminarily determined through a second derivative maximum point, when the first derivative corresponding to the Zuo Feng second derivative extreme point is smaller than or equal to 0, the extreme point is used as a left peak foot point, otherwise, the left search is continued until the first derivative is smaller than or equal to 0, and the point is used as the left peak foot point; when the first derivative corresponding to the extreme point of the second derivative of the right peak is greater than or equal to 0, the extreme point is taken as the right peak, otherwise, the search to the right is continued until the first derivative is greater than or equal to 0, and the point is taken as the right peak. Then, according to the characteristic inflection point obtained by searching, the peak position and peak height of each signal peak can be judged and obtained, the peak position and peak height are compared with a preset position interval and a peak height threshold value, the signal peak which is in the position interval and has the peak height exceeding the threshold value is screened out, and the subsequent secondary judgment processing is continued.
Specifically, the invention provides a secondary judging method for independent or multiple combination judgment by multiple analysis modes, which comprises peak height proportion analysis, peak area/peak height proportion analysis and mirror image proportion analysis. Wherein:
Peak height ratio analysis: in the preset position interval, the peak position of the peak signal P with the peak height exceeding the threshold value is x, and the peak height is h1. If the distance d exists, other peaks exist in the range [ x-d, x+d ], the peak height is h 2,h3,…,hn, the P is assumed to be a true target peak, the other peaks are assumed to be interference peaks, and the signal to noise ratio is calculated snr1=h1/h2,snr2=h1/h3,…,snrn-1=h1/hn.
Then, if snr_min=min { snr 1,snr2,…snrn-1 }. Ltoreq.n, n is a set threshold, and the value is 3-5 (specifically, the value is adjusted according to the running state of the instrument and the reagent), the peak P is an interference signal. The results of the unopened peak height ratio analysis are shown in fig. 6a, and the interfering signal is recognized as the target (16 yellow). Fig. 6 b shows the results after the on peak height ratio analysis, with the interfering signal identified and the target spot marked off.
Peak area/peak height ratio analysis: for a peak signal peak P with peak height exceeding a threshold value in a preset position interval, the peak height is h 1, the peak area is s 1, and if s 1/h1 is more than or equal to m, m is a set threshold value, the peak is an interference signal.
The m value is obtained by taking the corresponding optimum value (here, 6.1) through the distribution characteristic of the s/h ratio of the normal peak and the interference peak in the data as shown in fig. 7.
The results of the unopened peak area/peak height ratio analysis are shown in fig. 8 a, and the interfering signal is recognized as the target (68 yellow). Fig. 8 b shows the results after analysis of the ratio of the on peak area to the peak height, identifying the interfering signal and canceling the labeling of the target spot.
Mirror image proportion analysis: for a peak signal peak P with peak height exceeding a threshold value in a preset position interval, the point where the peak position is located is i, the position of a Zuo Feng foot inflection point is iL, and the position of a right peak foot inflection point is iR. d=min { i-iL, iR-i }, extracting the left peak signal yL of i-d-i, extracting the right peak signal yR of i-i+d, mirroring the yR signal with the i+d point to obtain yR ', calculating the correlation coefficient r based on yL and yR', and when r < rh, obtaining the peak as an interference signal. The optimal value of rh is 95% through data verification.
The result of the unopened mirror ratio analysis is shown in fig. 9 a, and the interfering signal is recognized as the target (82 yellow). Fig. 9 b shows the result of the open mirror ratio analysis, identifying the interfering signal and canceling the marking of the target spot.
On the basis of preprocessing the signals, the peak signals which are primarily marked as target targets are filtered for the second time through the analysis mode, so that the false positive problem caused by single peak height threshold judgment is avoided.
In summary, according to the signal processing and judging method based on capillary electrophoresis nucleic acid fragment analysis, a specific algorithm is set to carry out secondary identification according to the shape, the height and other characteristics of the signal peaks, so that accuracy of target spot judgment is greatly improved, experimenters are assisted in carrying out result judgment, and working efficiency is remarkably improved. The three different algorithms provided are used for identifying the signal peaks, and a user can select one or more algorithms to start according to actual conditions, so that high flexibility is provided.
The linear compensation of the amplitude of the peak type interval of the filtered signal eliminates the signal loss in the filtering process, thus ensuring the accuracy of the signal peak height and preventing the change of the real signal intensity caused by filtering.
It should be noted that all directional indicators (such as up, down, left, right, front, and rear … …) in the embodiments of the present invention are merely used to explain the relative positional relationship, movement, etc. between the components in a particular posture (as shown in the drawings), and if the particular posture is changed, the directional indicator is changed accordingly.
Furthermore, descriptions such as those referred to herein as "first," "second," "a," and the like are provided for descriptive purposes only and are not to be construed as indicating or implying a relative importance or an implicit indication of the number of features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In the description of the present invention, the meaning of "plurality" means at least two, for example, two, three, etc., unless specifically defined otherwise.
In the present invention, unless specifically stated and limited otherwise, the terms "connected," "affixed," and the like are to be construed broadly, and for example, "affixed" may be a fixed connection, a removable connection, or an integral body; can be mechanically or electrically connected; either directly or indirectly, through intermediaries, or both, may be in communication with each other or in interaction with each other, unless expressly defined otherwise. The specific meaning of the above terms in the present invention can be understood by those of ordinary skill in the art according to the specific circumstances.
In addition, the technical solutions of the embodiments of the present invention may be combined with each other, but it is necessary to be based on the fact that those skilled in the art can implement the technical solutions, and when the technical solutions are contradictory or cannot be implemented, the combination of the technical solutions should be considered as not existing, and not falling within the scope of protection claimed by the present invention.
Claims (9)
1. A method for signal processing and determination based on capillary electrophoresis nucleic acid fragment analysis, comprising the steps of:
S1: acquiring an original waveform sampling signal after capillary electrophoresis treatment after multiple polymerase chain reaction;
S2: preprocessing an original waveform sampling signal to obtain peak type characteristic point search after peak type optimization;
S3: performing preliminary screening on the signal peak target points under comparison judgment on the peak type characteristic points based on a preset position interval and a peak height threshold value;
s4: and (3) carrying out proportional combination analysis according to the characteristic points obtained by searching, and carrying out target marking under secondary judgment on the signal peaks based on analysis results.
2. The method for signal processing and determination based on capillary electrophoresis nucleic acid fragment analysis according to claim 1, wherein in said step S2, the pretreatment comprises baseline subtraction, peak height lossless filtering.
3. The method for signal processing and determination based on capillary electrophoresis nucleic acid fragment analysis according to claim 2, wherein said baseline subtraction uses a window sliding minimum extraction method for removing abnormal interference baseline signals.
4. The method for signal processing and determination based on capillary electrophoresis nucleic acid fragment analysis according to claim 2, wherein said peak height non-destructive filtering employs median filtering and polynomial smoothing for removing a hetero-peak.
5. The method for signal processing and determination based on capillary electrophoresis nucleic acid fragment analysis according to claim 1, wherein in said step S2, the peak-type feature point is a first derivative inflection point of the sampling signal.
6. The method for signal processing and determination based on capillary electrophoresis nucleic acid fragment analysis according to claim 1, wherein in said S4 step, the ratio combining analysis includes peak height ratio analysis, peak area/peak height ratio analysis and mirror image ratio analysis.
7. The method for signal processing and determination based on capillary electrophoresis nucleic acid fragment analysis according to claim 6, wherein said peak height ratio analysis is specifically:
And extracting the peak height of a target mark signal peak, comparing the peak height with the peak height in a preset distance range to obtain signal-to-noise ratio data, and if the signal-to-noise ratio data contains a signal-to-noise ratio smaller than a first set threshold value, judging the signal peak as an interference signal and canceling the target mark.
8. The method for signal processing and determination based on capillary electrophoresis nucleic acid fragment analysis according to claim 6, wherein said peak area/peak height ratio analysis is specifically:
and extracting peak height and peak area of a target mark signal peak, and judging the signal peak as an interference signal and canceling target mark if the ratio of the peak area to the peak height is larger than a second set threshold value.
9. The method for signal processing and determination based on capillary electrophoresis nucleic acid fragment analysis according to claim 6, wherein said image ratio analysis is specifically:
and taking the peak width of the target mark signal peak as an extraction range, taking the peak top position of the target mark signal peak as an extraction starting point, acquiring waveform signals of peaks in the extraction range around the extraction starting point, acquiring a correlation coefficient after mirror image processing, and judging the signal peak as an interference signal and canceling target mark if the correlation coefficient is smaller than a third set threshold value.
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