WO2018149114A1 - Method and device for determining microdeletion and microduplication in foetal chromosomes - Google Patents

Abstract

Disclosed are a method and device for determining microdeletion and microduplication in the foetal chromosomes. The method comprises the steps of: acquiring the concentration fm of fragments containing the microdeletion and microduplication; acquiring the male/female foetal nucleic acid concentration fy/fs; calculating the ratio rmY of the concentration fm of fragments containing the microdeletion and microduplication to the male foetal nucleic acid concentration fy, or the ratio rms of the fm to the female foetal nucleic acid concentration fs; calculating the rmY or rms according to the deleted copy number or the duplicated copy number, and filtering out false positives; taking the decimal part dmY of rmY or the decimal part dms of rms, determining whether dmY/dms is positive or not, and if not, then filtering out the result; calculating the sum amY of the concentration fm of fragments containing the microdeletion and microduplication and the male foetal nucleic acid concentration fy, or the sum ams of the fm and the female foetal nucleic acid concentration fs; and filtering the microdeletion and microduplication fragments according to a judgement principle, and obtaining the microdeletion and microduplication fragments of the foetal chromosomes after filtering.

Description

Method and device for determining microdeletion microduplication in fetal chromosome Technical field

The present invention relates to the field of biomedicine, and in particular, to methods and apparatus for determining microdeletions and microduplications in chromosomes.

Background technique

In the non-invasive prenatal testing (NIPT) clinical field, screening for fetal microdeletion microrepetition is less sensitive. Existing methods for estimating fetal microdeletion microduplication based on maternal peripheral blood are mainly divided into the following two directions: 1) Estimating microdeletion microduplication based on changes in the proportion of embryonic DNA sequences in maternal plasma. 2) Using the differentiation of single nucleotide polymorphism (SNPs) locus expression, multiple SNP loci were selected for estimation.

The existing detection methods have certain limitations. The detection method 1) has low precision, and a large number of false positive results will occur. Since the results are only based on the change of the proportion of fragments in a certain region, the detection results are lacking, and the effective filtering is lacking. Method, the appearance of false positives is difficult to avoid. Method 2) requires probe capture and high-depth sequencing, or needs to obtain parent-source information. High-depth capture requires design of the chip, which increases the difficulty of the experiment. High-depth sequencing increases the cost, and the uncaptured part cannot. Determination.

Summary of the invention

It is an object of the present invention to provide a method and apparatus for determining microdeletion microduplication in a fetal chromosome by evaluating the microdeletion microrepetition by calculating the concentration of the microdeletion of the fetal microdeletion and the concentration of the nucleic acid of the fetus itself. False positive rate, high precision.

Based on the above objects, an aspect of the present invention provides a method for determining a micro defect in a fetal chromosome The method of losing duplicates includes the following steps:

S1, obtaining a concentration fm containing a microdeletion microrepeat fragment;

S2, obtaining a male fetal nucleic acid concentration fy or a female fetal nucleic acid concentration fs;

S3. Calculating a ratio rmY=fm/fy of the concentration fm containing the microdeletion microrepeat fragment to the male fetal nucleic acid concentration fy, or calculating a ratio of the concentration fm containing the microdeletion microrepeat fragment to the female fetal nucleic acid concentration fs rms=fm/fs ;

S4, calculating rmY or rms according to the missing copy number or the repeated copy number, filtering out the false positive;

S5, taking the fractional part dmY of rmY, or the fractional part dms of rms, determining whether dmY or dms is positive, otherwise filtering out the result;

S6. Calculating the sum of the concentration fm containing the microdeletion microrepeat fragment and the male fetal nucleic acid concentration fy is amY=fm+fy, or calculating the sum of the concentration fm containing the microdeletion microrepeat fragment and the female fetal nucleic acid concentration fs is ams=fm +fs;

S7, filtering the micro-deletion micro-repeat fragments according to the judgment principle, and filtering to obtain a fetal chromosome micro-deletion micro-repeat fragment.

Another aspect of the invention also provides an apparatus for determining microdeletion microrepetitions in a fetal chromosome, comprising:

a micro-deletion micro-repeat fragment concentration calculating device for obtaining a concentration fm containing a micro-deletion micro-repeat fragment;

a fetal nucleic acid concentration obtaining device for obtaining a male fetal nucleic acid concentration fy or a female fetal nucleic acid concentration fs;

a ratio calculating device for calculating a ratio rmY=fm/fy of a concentration fm containing a microdeletion microrepetition fragment to a male fetal nucleic acid concentration fy, or calculating a ratio rms of a concentration fm containing a microdeletion microrepeat fragment to a female fetal nucleic acid concentration fs =fm/fs;

a first filtering device for calculating rmY or rms according to the missing copy number or repeated copy number, filtering out false positives;

a second filtering device for taking the fractional part of the rmY dmY, or the fractional part of the rms Divide dms to determine whether dmY or dms is positive, otherwise the result is filtered out;

And a value calculation device for calculating the sum of the concentration fm containing the microdeletion microrepetition fragment and the male fetal nucleic acid concentration fy as amY=fm+fy, or calculating the concentration fm containing the microdeletion microrepeat fragment and the female fetal nucleic acid concentration fs And ams=fm+fs;

The third filtering device is configured to filter the micro-deletion micro-repeat fragments according to the determination principle, and filter to obtain a fetal chromosome micro-deletion micro-repeat fragment.

The method and device provided by the invention can accurately determine the microdeletion microrepetition in the chromosome, and is particularly suitable for determining the microdeletion microrepetition of the fetal chromosome in the peripheral blood of the pregnant woman.

Compared with the prior art:

1. The invention does not require an additional chip design, saves the cost of the chip design, and makes the experimental method simple.

2. Without high-level sequencing, the subsequent processing of the data on the basis of chromosome aneuploidy using whole genome data can directly yield accurate micro-deletion micro-repetition results without increasing the amount of data.

3. Overcoming the prior art method using snp may miss some areas that cannot be captured, and the present invention can be detected on the whole genome.

4. For the first time, the whole genome sequencing method was used to determine the micro-repetition concentration of fetal microdeletions.

5. Reduced false positives and improved accuracy.

6. Remove the influence of the micro-repetition of the mother micro-deletion on the fetus and improve the accuracy.

DRAWINGS

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a flow diagram of a method of determining fetal microdeletion microduplication in an embodiment of the invention.

Figure 2 is a flow diagram of a method of obtaining a concentration fm of a fragment containing a microdeletion microrepeat in the embodiment of Figure 1.

3 is a flow chart of a method of obtaining a micro-deletion micro-repeat final window in the embodiment of FIG. 1.

4 is a flow chart of a method of obtaining a male fetal nucleic acid concentration fy in the embodiment of FIG. 1.

Figure 5 is a flow diagram of a method of obtaining a female fetal nucleic acid concentration fs in the embodiment of Figure 1.

Figure 6 is a flow chart of a method of obtaining a predetermined range in the method of Figure 5.

7 is a flow chart of a method of obtaining a predetermined function in the method of FIG. 5.

Figure 8 is a block diagram showing the structure of an apparatus for determining microdeletion microduplication in a fetal chromosome in another embodiment of the present invention.

Fig. 9 is a block diagram showing the configuration of a concentration calculating apparatus containing microdeletion microrepetition fragments in the embodiment of Fig. 8.

FIG. 10 is a structural block diagram of an ultimate window obtaining unit in which the micro-deletion micro-repetition in the embodiment of FIG. 8 is located.

Figure 11 is a block diagram showing the structure of a male fetal nucleic acid concentration fy obtaining unit in the embodiment of Figure 8.

Figure 12 is a block diagram showing the structure of a female fetal nucleic acid concentration fs obtaining unit in the embodiment of Figure 8.

Figure 13 is a block diagram showing the structure of a predetermined range determining element in the embodiment of Figure 8.

Figure 14 is a block diagram showing the structure of a predetermined function determining element in the embodiment of Figure 8.

Figure 15 is a graph showing the results of 19 sample microdeletion microrepeats in Example 2.

Main component symbol description

Equipment for determining microdeletion and microduplication in fetal chromosomes 100 Microdeletion micro-repeat fragment concentration calculation device 110 Primal window acquisition unit 111 First average depth obtaining unit 112

Second average depth obtaining unit 113 Microdeletion microrepeat fragment concentration obtaining unit 114 The micro-missing micro-repetition is the ultimate window obtaining unit 115 First sequencing component 1151 Alignment component 1152 Length determining component 1153 Primary window determination component 1154 First statistical component 1155 Correction component 1156 First merge component 1157 First filter element 1158 Second merge component 1159 Repetitive component 1160 Microdeletion microrepetition ultimate window determining component 1161 Fetal nucleic acid concentration obtaining device 120 Male fetal nucleic acid concentration fy obtaining unit 121 Second sequencing component 1211 First number determining component 1212 Filter module 12121 Second statistical component 1213 Average depth acquisition component 1214 Male fetal nucleic acid concentration acquisition component 1215 Female fetal nucleic acid concentration fs obtaining unit 122 Third sequencing component 1221 Second number determining component 1222 Frequency determining component 1223

Female fetal nucleic acid concentration determining element 1224 Predetermined range determining component 1225 Length determination module 12251 First frequency determination module 12252 Correlation coefficient determination module 12253 Scheduled range determination module 12254 Predetermined function determining component 1226 Second frequency determination module 12261 Fitting module 12262 Ratio calculation device 130 First filter device 140 False positive judgment unit 141 Second filter device 150 Positive judgment unit 151 And value calculation device 160 Third filter device 170

The invention will be further illustrated by the following detailed description in conjunction with the accompanying drawings.

detailed description

Embodiments of the present invention are described in detail below. The embodiments described below with reference to the accompanying drawings are intended to be illustrative of the invention and are not to be construed as limiting.

It should be noted that the terms "primary", "secondary", "ultimate" are used for descriptive purposes only, and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, features defining "primary", "secondary", "ultimate" may include one or more of the features, either explicitly or implicitly. Further, in the description of the present invention, the meaning of "a plurality" is two or more unless otherwise specified. This hair The "unique alignment sequence" and "unique alignment sequence" in the Ming are sometimes referred to as "sequences" and "sequencing sequences".

The term "parent sample" refers herein to a biological sample obtained from a pregnant subject, eg, a woman.

The term "microdeletion microrepetition" refers to deletions or duplications on the chromosome that range from 1.5 kb to 10 Mb in length.

The term "GC correction" refers to the correction of the GC content in the sequence.

Referring to Figure 1, the present invention provides a method for determining microduplication of fetal chromosomal microdeletions, comprising:

S1, obtaining a concentration fm containing a microdeletion microrepeat fragment;

S2, obtaining a male fetal nucleic acid concentration fy or a female fetal nucleic acid concentration fs;

S3. Calculating a ratio rmY=fm/fy of the concentration fm containing the microdeletion microrepeat fragment to the male fetal nucleic acid concentration fy, or calculating a ratio of the concentration fm containing the microdeletion microrepeat fragment to the female fetal nucleic acid concentration fs rms=fm/fs ;

S4, calculating rmY or rms according to the missing copy number or the repeated copy number, filtering out the false positive;

S5, taking the fractional part dmY of rmY, or the fractional part dms of rms, determining whether dmY or dms is positive, otherwise filtering out the result;

S6. Calculating the sum of the concentration fm containing the microdeletion microrepeat fragment and the male fetal nucleic acid concentration fy is amY=fm+fy, or calculating the sum of the concentration fm containing the microdeletion microrepeat fragment and the female fetal nucleic acid concentration fs is ams=fm +fs;

S7, filtering the micro-deletion micro-repeat fragments according to the judgment principle, and filtering to obtain a fetal chromosome micro-deletion micro-repeat fragment.

The inventors have surprisingly found that the method of the present invention enables accurate determination of microdeletion microrepetitions in chromosomes, and is particularly useful for determining fetal chromosomes in peripheral blood of pregnant women. Microdeletion microrepetition.

Referring to Fig. 2, according to an embodiment of the present invention, the concentration fm of the microdeletion microrepetition fragment in the step S1 is obtained by the following steps:

S11. Obtaining a primary window containing no microdeletion microrepetition according to a primary window containing microdeletion microrepetitions, calculating a total number of primary windows containing microdeletion microrepetitions and a total number of primary windows containing microdeletion microrepetitions, and The total number of sequences of primary windows that do not contain microdeletion microduplications and the total number of primary windows that do not contain microdeletion microduplications;

S12, obtaining an average depth d1 of the primary window containing the microdeletion microrepetition, d1=the total number of sequences of the primary window containing the microdeletion microrepetitions/the total number of primary windows containing the microdeletion microrepetitions;

S13, obtaining an average depth d2 of the primary window containing no microdeletion microrepetition, d2 = total number of primary windows without microdeletion microrepetitions / total number of primary windows without microdeletion microrepetitions;

S14. Calculating a concentration fm containing microdeletion microrepeats, fm=2×-d2-d1-/d2.

As will be understood by those skilled in the art, the total number of primary windows and the number of sequences without the microdeletion microrepetition can be derived from a method containing a microdeletion microrepeat end window. For example, the final window has an absolute coordinate of the start and end positions. Based on the coordinates of the secondary window, the coordinates of the secondary window are found, and then it is confirmed how many primary windows are in the secondary window, and the initial and final primary windows are removed. To exclude fluctuations in the data, and then get the final primary window, calculate the total number of sequences.

Referring to Figure 3, in accordance with an embodiment of the present invention, the final window containing microdeletion microduplication is obtained by the following steps:

S111. Perform nucleic acid sequencing on a biological sample containing free nucleic acid to obtain a sequencing result composed of a plurality of sequencing data;

S112, aligning the sequencing result with a reference genome to construct a unique alignment sequencing sequence set, each of the unique alignment sequencing sequence sets can only match one position of the reference genome;

S113. Determine a length of each unique alignment sequencing sequence in the unique alignment sequencing sequence set;

S114, dividing the reference genome into a plurality of primary windows according to a predetermined length, the predetermined length being 1 bp-5M;

S115. Count the number of each unique alignment sequence that falls into each primary window;

S116: performing GC correction on the number of sequences falling in the primary window, and performing batch-to-batch adjustment on the corrected result;

S117. Combine a predetermined number of adjacent primary windows into a plurality of secondary windows, and determine a number of sequences in each secondary window.

S118, performing statistical tests on each secondary window, calculating a T1 value, and filtering the secondary window according to the T1 value;

S119. Perform a statistical test on the filtered secondary window, calculate a T2 value, and merge two adjacent secondary windows having no significant difference into an ultimate window according to the T2 value;

S120, repeat steps S118-S120 until they cannot be merged;

S121: Perform a hypothesis test on the final window obtained by the final combination, and obtain an ultimate window containing the micro-deletion micro-repeat.

According to an embodiment of the invention, the biological sample containing the free nucleic acid is free fetal nucleic acid in the peripheral blood of the pregnant woman.

According to an embodiment of the invention, the nucleic acid is DNA.

According to an embodiment of the invention, the sequencing result comprises a length of the free nucleic acid and a base arrangement order. The "length" refers to the length of the nucleic acid, and can be expressed in units of base pairs, that is, bp.

According to an embodiment of the invention, the sequencing is double-end sequencing, single-end sequencing or single-molecule sequencing. Thereby, the length of the free nucleic acid is easily obtained, which is advantageous for the subsequent steps.

It will be understood by those skilled in the art that since the free fetal DNA in the blood sample is relatively short, it is necessary to obtain the length of all the free DNA molecules, so that single-end sequencing requires measurement of the entire free DNA molecule, or double-end sequencing.

According to an embodiment of the present invention, the predetermined length in the step S114 is 1 bp to 5 M, and the predetermined number in the step S117 is 5 to 100. Preferably, the predetermined length is 20-40 Kb.

According to an embodiment of the invention, the method of GC correction comprises using local weighted regression, linear regression or logistic regression.

According to one embodiment of the invention, the inter-batch adjustment is to calculate a baseline for each primary window corresponding to all samples in the sequence of the sequencing, based on the number of unique alignment sequencing sequences within each primary window based on the baseline. Weighted correction.

According to an embodiment of the invention, the value of T1 in the step S118 comprises calculating according to a Z-test or a T-test, the filtering is filtering out the secondary window in which the T1 value is between -3-3.

According to an embodiment of the present invention, the value of T2 in the step S119 is calculated according to a rank sum test, a symbol test or a run test, and the non-significant difference is that the T2 value of the adjacent two windows is -3-3. between.

According to an embodiment of the invention, the hypothesis test in the step S121 comprises calculating according to a Z test or a T test, the test threshold being defined as 3. That is, when the statistic of the test is >3 or <-3, it is determined to be the final window containing the microdeletion microrepetition.

Referring to FIG. 4, according to an embodiment of the present invention, the male fetal nucleic acid concentration fy in the step S2 is obtained by the following steps:

S211, sequencing a biological sample containing free nucleic acid to obtain a sequencing result composed of a plurality of sequencing data;

S212. Determine, according to the sequencing result, a number of unique alignment sequences in the Y chromosome in the sample that fall into the primary window;

S213. Count the sum of the number of unique alignment sequencing sequences in each primary window on the Y chromosome and the total number of the primary windows;

S214, obtaining an average depth dy of the primary window in the Y chromosome, dy=the sum of the number of unique aligned sequencing sequences on the Y chromosome/the number of primary windows on the Y chromosome;

S215, obtaining a male fetal nucleic acid concentration fy, fy=2×dy/d2, the d2 is the average depth of the primary window without the microdeletion microrepetition, and d2=the total number of primary windows without the microdeletion microrepetition/ The number of primary windows that do not contain microdeletion microduplication.

As will be understood by those skilled in the art, the total number of primary windows and the number of sequences without the microdeletion microrepetition can be derived from a method containing a microdeletion microrepeat end window.

According to an embodiment of the present invention, the step S212 further comprises: dividing the reference genome into a plurality of primary windows according to a predetermined length, and removing the primary window in the Y chromosome whose number of unique alignment sequences is more than 5 times the number of the average sequence. Preferably, the primary window is a primary window adjusted by GC modification.

Referring to FIG. 5, according to an embodiment of the present invention, the female fetal nucleic acid concentration fs in the step S2 is obtained by the following steps:

S221, sequencing a biological sample containing free nucleic acid to obtain a sequencing result composed of a plurality of sequencing data;

S222. Determine, according to the sequencing result, a number of unique alignment sequencing sequences whose length falls within a predetermined range in the sample;

S223. The number of unique alignment sequencing sequences falling within a predetermined range based on the length Determining a frequency at which a unique alignment sequence occurs within the predetermined range;

S224. Determine a female fetal nucleic acid concentration fs in the sample according to a predetermined function according to a frequency at which the unique alignment sequence appears within the predetermined range.

Referring to FIG. 6, according to an embodiment of the present invention, the predetermined range in the step S222 is determined by the following steps:

S2221. Determine a length of the unique alignment sequencing sequence included in the plurality of control samples;

S2222: setting a plurality of candidate length ranges, and respectively determining a frequency of the unique alignment sequencing sequence that appears in each candidate length range of the plurality of control samples;

S2223: determining a frequency of each of the candidate length ranges and a concentration of the nucleic acid in the control sample based on a frequency of the unique alignment sequence and a concentration of the nucleic acid in the control sample in each candidate length range based on the plurality of control samples. Correlation coefficient

S2224. Determine, according to the value of the correlation coefficient, at least one candidate length range or a candidate length range combination as the predetermined range.

According to an embodiment of the invention, the predetermined range is determined based on a plurality of control samples, wherein the concentration of the nucleic acid in the control sample is known, preferably, the predetermined range is based on at least 20 control samples definite.

According to an embodiment of the invention, the control sample is a maternal peripheral blood sample of a normal male fetus with a known ratio of free fetal nucleic acid, and the nucleic acid concentration in the control sample is determined using the Y chromosome.

According to one embodiment of the invention, the free fetal nucleic acid concentration in the control sample is determined using the Y chromosome, i.e., by the method of the above-described male fetal nucleic acid concentration fy of the present invention.

According to an embodiment of the present invention, the candidate length range in the S2222 spans from 1 to 300 bp, preferably from 1 to 20 bp.

According to an embodiment of the invention, the plurality of candidate length ranges have a step size of 1-2 bp.

For example, the candidate length ranges are 1-20, 2-21,3-22, ..., respectively, wherein the span is 20 bp and the step size is 1 bp.

According to an embodiment of the present invention, the predetermined range in the step S222 is 179 bp to 206 bp.

Referring to FIG. 7, according to an embodiment of the present invention, the predetermined function in the step S223 is obtained by the following steps:

S2231, respectively, determining, in the plurality of control samples, a frequency at which a unique alignment sequencing sequence occurs within the predetermined range;

S2232: Fitting a frequency of the unique alignment sequencing sequence within the predetermined range among the plurality of control samples with a known nucleic acid concentration to determine the predetermined function.

According to an embodiment of the invention, the fit is a linear fit.

According to an embodiment of the invention, the predetermined function is d = -0.3215 x p + 1.62562, wherein d represents the nucleic acid concentration and p represents the frequency of the unique aligned sequencing sequence occurring within the predetermined range.

According to an embodiment of the present invention, the step S4 further includes: if the rmY≧2 is calculated according to the missing copy number or the repeated copy number is calculated to obtain rmY≧6, it is determined to be untrustworthy, and the fake is filtered. Positive result

Alternatively, if rms≧2 is calculated based on the missing copy number or the repeated copy number is calculated to obtain rms≧6, it is determined to be unreliable, and the false positive result is filtered out.

According to an embodiment of the present invention, the step S5 further comprises: if dmY<0.13 or dmY>0.85, dmY is positive;

Alternatively, if dms < 0.15 or dms > 0.791, dms is positive.

According to an embodiment of the present invention, the determining principle in the step S7 is: if amY is between 0.95 and 1.05, the fragment of the microdeletion microrepetition is considered to be from the mother, and the fragment of the microdeletion microrepetition is filtered. ;

Alternatively, if ams is between 0.93-1.06, the microdeletion microrepetitive fragment is considered to be from the mother, and the microdeletion microrepetitive fragment is filtered.

Referring to Figure 8, an aspect of the present invention also provides an apparatus 100 for determining microdeletion microrepetitions in a fetal chromosome, comprising:

a micro-deletion micro-repeat fragment concentration calculating device 110 for obtaining a concentration fm containing a micro-deletion micro-repeat fragment;

a fetal nucleic acid concentration obtaining device 120 for obtaining a male fetal nucleic acid concentration fy or a female fetal nucleic acid concentration fs;

The ratio calculating means 130 is configured to calculate a ratio rmY=fm/fy of the concentration fm containing the microdeletion microrepetition fragment to the male fetal nucleic acid concentration fy, or calculate a ratio of the concentration fm containing the microdeletion microrepeat fragment to the female fetal nucleic acid concentration fs Rms=fm/fs;

a first filtering device 140, configured to calculate rmY or rms according to the missing copy number or repeated copy number, and filter out false positives;

The second filtering device 150 is configured to take the fractional part dmY of rmY, or the fractional part dms of rms, to determine whether dmY or dms is positive, or filter out the result;

And a value calculation device 160 for calculating the sum of the concentration fm containing the microdeletion microrepetition fragment and the male fetal nucleic acid concentration fy as amY=fm+fy, or calculating the concentration fm containing the microdeletion microrepeat fragment and the female fetal nucleic acid concentration fs The sum of ams=fm+fs;

The third filtering device 170 is configured to filter the micro-deletion micro-repeat fragments according to the determination principle, and filter to obtain a fetal chromosome micro-deletion micro-repeat fragment.

Referring to FIG. 9, according to an embodiment of the present invention, the micro-deletion micro-repeat fragment concentration calculating apparatus 110 further includes:

An initial pole window obtaining unit 111, configured to obtain a primary window containing no microdeletion microrepetition according to a primary window containing microdeletion microrepetitions, calculate a total sequence number of primary windows containing microdeletion microduplications, and microdeletion microduplications The total number of primary windows, as well as the total number of sequences of primary windows without microdeletion microduplications and the number of primary windows that do not contain microdeletion microduplications;

a first average depth obtaining unit 112 for obtaining an average depth d1 of the primary window containing the microdeletion microrepetition, d1=the total number of sequences of the primary window containing the microdeletion microrepetitions/the number of primary windows containing the microdeletion microrepetitions;

a second average depth obtaining unit 113 for obtaining an average depth d2 of the primary window without the microdeletion microrepetition, d2=the total number of sequences of the primary window without the microdeletion microrepetition/the primary window without the microdeletion microrepetition Total number of;

The microdeletion micro-repulsion fragment concentration obtaining unit 114 is configured to calculate the concentration fm containing the micro-deletion micro-repeat fragment, fm=2×-d2-d1-/d2.

According to an embodiment of the present invention, the micro-deletion micro-repetition fragment concentration calculation device 110 further includes a micro-deletion micro-repetition in the ultimate window obtaining unit 115. Referring to FIG. 10, the micro-deletion micro-repetition in the final window obtaining unit 115 includes:

a first sequencing component 1151 for performing nucleic acid sequencing on a biological sample containing free nucleic acid to obtain a sequencing result composed of a plurality of sequencing data;

Aligning element 1152 for aligning the sequencing result with a reference genome to construct a unique alignment sequencing sequence set, each of the unique alignment sequencing sequence sets being capable of only one of the reference genomes Position matching

a length determining component 1153 for determining a length of each unique aligned sequencing sequence in the unique aligned sequencing sequence set;

a primary window determining component 1154 for dividing the reference genome into a plurality of primary windows according to a predetermined length, the predetermined length being 1 bp - 5 M;

a first statistical component 1155, configured to count the number of each unique alignment sequencing sequence falling into each primary window;

a correction component 1156 for performing GC correction on the number of sequences falling into the primary window and performing inter-batch adjustment on the corrected result;

a first merging element 1157, configured to combine a predetermined number of adjacent primary windows into a plurality of secondary windows, and determine a number of sequences in each secondary window;

a first filter element 1158, configured to perform statistical verification on each secondary window, calculate a T1 value, and filter the secondary window according to the T1 value;

a second merging component 1159 is configured to perform a statistical check on the filtered secondary window, calculate a T2 value, and merge two adjacent secondary windows having no significant difference into an ultimate window according to the T2 value;

Repeating element 1160 for repeatedly starting the first filter element 1158 and the second combining element 1159 until it cannot be merged;

The micro-deletion micro-repeat final window determining component 1161 is configured to perform a hypothesis test on the final merged final window to obtain an ultimate window containing micro-deletion micro-repeats.

According to an embodiment of the invention, the predetermined number of the first merging elements 1157 is between 5 and 100. Preferably, the predetermined length is 20-40 Kb.

According to one embodiment of the invention, the method of GC correction in the correction element 1156 includes the use of local weighted regression, linear regression or logistic regression.

According to one embodiment of the invention, the inter-batch adjustment in the correction element 1156 is to calculate a baseline for each primary window corresponding to all samples in the sequenced batch, a unique alignment within each primary window based on the baseline. The number of sequencing sequences is weighted and corrected.

According to an embodiment of the invention, the T1 value in the first filter element 1158 comprises a calculation based on a Z-test or a T-test, which filter is filtered out of a secondary window having a T1 value between -3-3.

According to an embodiment of the invention, the T2 value in the second combining element 1159 is calculated according to a rank sum test, a symbol check or a run test, the insignificant difference being the T2 value of the adjacent two windows at -3 Between -3.

According to an embodiment of the invention, the hypothesis test in the micro-deletion micro-repeat final window determining element 1161 comprises calculating from a Z-test or a T-test, the test threshold being defined as three. That is, when the statistic of the test is >3 or <-3, it is determined to be the final window containing the microdeletion microrepetition.

According to an embodiment of the present invention, the fetal nucleic acid concentration obtaining unit 121 further includes a male fetal nucleic acid concentration fy obtaining unit 121. Referring to FIG. 11, the male fetal nucleic acid concentration fy obtaining unit 121 includes:

a second sequencing component 1211 for sequencing a biological sample containing free nucleic acid to obtain a sequencing result composed of a plurality of sequencing data;

a first number determining component 1212, configured to determine, according to the sequencing result, a number of unique aligned sequencing sequences in the Y chromosome in the sample that fall within a primary window;

a second statistical component 1213 for counting the sum of the number of unique aligned sequencing sequences in each primary window on the Y chromosome and the total number of primary windows;

The average depth obtaining element 1214 is for obtaining the average depth dy of the primary window in the Y chromosome, dy=the sum of the number of unique aligned sequencing sequences on the Y chromosome/the number of primary windows on the Y chromosome;

Male fetal nucleic acid concentration obtaining element 1215 for obtaining male fetal nucleic acid concentration fy, fy = 2 x dy / d2, said d2 is the average depth of the primary window without microdeletion microrepetition, d2 = no microdeletion microrepetition The total number of sequences in the primary window / the number of primary windows without micro-deletion micro-repeats.

According to an embodiment of the invention, the first number determining element 1212 further comprises a filtering module 12121 for grouping reference genes by a predetermined length Divided into a plurality of primary windows, the primary window in which the number of unique alignment sequences in the Y chromosome is more than 5 times larger than the average number of sequences is removed.

According to an embodiment of the present invention, the fetal nucleic acid concentration obtaining device 120 further includes a female fetal nucleic acid concentration fs obtaining unit 122. Referring to FIG. 12, the female fetal nucleic acid concentration fs obtaining unit 122 includes:

a third sequencing component 1221, configured to sequence a biological sample containing free nucleic acid to obtain a sequencing result composed of a plurality of sequencing data;

a second number determining component 1222, configured to determine, according to the sequencing result, a number of unique aligned sequencing sequences whose length falls within a predetermined range in the sample;

a frequency determining component 1223 for determining a frequency at which a unique alignment sequencing sequence occurs within the predetermined range based on the number of unique aligned sequencing sequences whose length falls within a predetermined range;

A female fetal nucleic acid concentration determining element 1224 is configured to determine a female fetal nucleic acid concentration fs in the sample according to a predetermined function based on a frequency of the unique aligned sequencing sequence occurring within the predetermined range.

According to an embodiment of the present invention, the female fetal nucleic acid concentration fs obtaining unit 122 further includes a predetermined range determining component 1225. Referring to FIG. 13, according to an embodiment of the present invention, the predetermined range determining component 1225 further includes:

a length determining module 12251, configured to determine a length of a unique alignment sequencing sequence included in the plurality of control samples;

The first frequency determining module 12252 is configured to set a plurality of candidate length ranges, and respectively determine a frequency of the unique aligned sequencing sequence that occurs in each candidate length range of the plurality of control samples;

a correlation coefficient determination module 12253, configured to generate a frequency of uniquely aligned sequencing sequences within each candidate length range based on the plurality of control samples, and in the control sample a concentration of the nucleic acid, determining a correlation coefficient between each of the candidate length ranges and a concentration of the nucleic acid in the control sample;

The predetermined range determining module 12254 is configured to determine, according to the value of the correlation coefficient, at least one candidate length range or a candidate length range combination as the predetermined range.

According to an embodiment of the invention, the predetermined range is determined based on a plurality of control samples, wherein the nucleic acid concentration in the control sample is known, preferably, the predetermined range is determined based on at least 20 control samples of.

According to an embodiment of the invention, the control sample is a maternal peripheral blood sample of a normal male fetus with a known ratio of free fetal nucleic acid, and the free fetal nucleic acid concentration in the control sample is determined using the Y chromosome. That is, it is determined by the method of the above-described male fetal nucleic acid concentration fy of the present invention.

According to an embodiment of the invention, the candidate length range spans from 1 to 300 bp, preferably from 1 to 20 bp.

According to an embodiment of the invention, the plurality of candidate length ranges have a step size of 1-2 bp.

For example, the candidate length ranges are 1-20, 2-21,3-22, ..., respectively, wherein the span is 20 bp and the step size is 1 bp.

According to an embodiment of the invention, the predetermined range is from 179 bp to 206 bp.

According to an embodiment of the present invention, the female fetal nucleic acid concentration fs obtaining unit 122 further includes a predetermined function determining component 1226. Referring to FIG. 14, the predetermined function determining component 1226 includes:

a second frequency determining module 12261, configured to determine, in the plurality of control samples, a frequency of occurrence of a unique alignment sequencing sequence within the predetermined range, respectively;

a fitting module 12262 for using the plurality of control samples in the predetermined range The frequency at which a unique aligned sequencing sequence occurs is fitted to a known nucleic acid concentration to determine the predetermined function.

According to an embodiment of the invention, the fit is a linear fit.

According to an embodiment of the invention, the predetermined function is d = -0.3215 x p + 1.62562, wherein d represents the free fetal nucleic acid concentration and p represents the frequency at which the unique aligned sequencing sequence occurs within the predetermined range.

According to an embodiment of the present invention, the first filtering device 140 further includes a false positive determining unit 141, configured to calculate rmY≧6 if rmY≧2 or the repeated copy number is calculated according to the missing copy number. , then judged to be untrustworthy, filtering out false positive results;

Alternatively, if rms≧2 is calculated based on the missing copy number or the repeated copy number is calculated to obtain rms≧6, it is determined to be unreliable, and the false positive result is filtered out.

According to an embodiment of the present invention, the second filtering device 150 further includes a positive determining unit 151 for determining that dmY is positive if dmY<0.13 or dmY>0.85; or, if dms<0.15 or dms>0.791, Then dms is positive.

According to an embodiment of the present invention, the determining principle in the third filtering device 170 is: if amY is between 0.95 and 1.05, the fragment of the microdeletion microrepetition is considered to be from the mother, and the microdeletion is filtered. Repeated segment

Alternatively, if ams is between 0.93-1.06, the microdeletion microrepetitive fragment is considered to be from the mother, and the microdeletion microrepetitive fragment is filtered.

Example 1

First, obtaining the concentration fm of the microdeletion microrepetitive fragment;

1. Performing nucleic acid sequencing on a biological sample containing free nucleic acid to obtain a sequencing result composed of a plurality of sequencing data;

2. Aligning the sequencing results with a reference genome to construct a unique aligned sequencing sequence set, each of the unique aligned sequencing sequences being capable of only matching one position of the reference genome;

3. determining the length of each unique alignment sequencing sequence in the unique alignment sequencing sequence set;

4. The reference genome is divided into a plurality of primary windows according to a predetermined length, the predetermined length being 1 bp to 5 M, preferably 20 kp to 40 kp being a predetermined length, for example (1-20 bp, 20-40 bp, 40-80 bp, 80) -100bp, 100-120bp, ...,);

5. Counting the number of unique aligned sequencing sequences in which each of the unique aligned sequencing sequences falls within each primary window;

6. Perform GC correction on the number of sequences falling into the primary window, and perform batch-to-batch adjustment on the corrected result, including the method of local weighted regression, linear regression or logistic regression;

7. Combining a predetermined number of adjacent primary windows into a plurality of secondary windows, determining the number of sequences in each secondary window, the predetermined number being 5-100; for example, combining 5 primary windows into 1 time The first window is 1-20 bp, 20-40 bp, 40-80 bp, 80-100 bp, 100-120 bp, and the combined secondary window is 1-120 bp.

8. Perform a statistical test on each secondary window to calculate a T1 value, which is calculated by a Z test or a T test;

9. Filtering the secondary window according to the T1 value, that is, filtering the secondary window whose T1 value is between -3-3;

10. Perform a statistical test on the filtered secondary window to calculate a T2 value including, but not limited to, calculated according to a rank sum test, a symbol test, or a run test;

11. Combine two adjacent secondary windows with no significant difference according to the T2 value. a pole window, the insignificant difference is that the T2 of the two windows is between -3-3;

12. Repeat 8-10 until you cannot merge;

13. Perform a hypothesis test on the final window obtained by the final combination, and obtain an ultimate window containing micro-deletion micro-repetitions, which is calculated according to a Z-test or a T-test, that is, when the statistic of the test is >3 or <-3, It was judged to be the ultimate window containing microdeletion microduplication.

14. Obtaining a primary window containing no microdeletion microrepetitions based on a primary window containing microdeletion microrepetitions, calculating a total number of primary windows containing microdeletion microrepetitions and a total number of primary windows containing microdeletion microduplications, and The total number of sequences of primary windows that do not contain microdeletion microduplications and the total number of primary windows that do not contain microdeletion microduplications;

15. Calculating the average depth d1 of the final window containing microdeletion microrepetitions, d1 = total number of sequences of primary windows containing microdeletion microduplications / total number of final windows containing microdeletion microduplications;

16. Calculating the average depth d2 of the primary window without microdeletion microrepetitions, d2 = total number of primary windows without microdeletion microduplications / total number of primary windows without microdeletion microduplications;

17. Calculate the concentration fm of the microdeletion microrepetitive fragment, fm = 2 x - d2 - d1 - / d2.

2. Obtaining a male fetal nucleic acid concentration fy or a female fetal nucleic acid concentration fs;

1. Determine whether the Y chromosome is contained in the sample to be tested, and if so, calculate the male fetal nucleic acid concentration fy, if not, calculate the female fetal nucleic acid concentration fs;

2. If the Y chromosome is contained, the male fetal nucleic acid concentration fy is calculated.

(1) determining, according to the above sequencing result, the number of unique alignment sequences in the Y chromosome in the sample falling into the primary window;

(2) Remove the large number of unique alignment sequences adjusted by GC modification in the primary window a primary window that is more than 5 times the average number of sequences;

(3) counting the sum of the number of unique alignment sequencing sequences in each primary window on the Y chromosome and the total number of primary windows;

(4) Obtain the average depth dy of the primary window in the Y chromosome, dy=the sum of the number of unique alignment sequences on the Y chromosome/the number of primary windows on the Y chromosome;

(5) Obtaining a male fetal nucleic acid concentration fy,fy=2×dy/d2, the d2 is the average depth of the primary window without the microdeletion microrepetition, and d2=the total number of primary windows without the microdeletion microrepetition / Number of primary windows without micro-deletion micro-repeats.

3. If the Y chromosome is not included, calculate the female fetal nucleic acid concentration fs.

(1) determining the number of unique alignment sequencing sequences in which the length of the biological sample containing the free nucleic acid falls within a predetermined range; the predetermined range is 179 bp to 206 bp.

The predetermined range is obtained by the following steps:

a, selecting at least 20 control samples, ie samples containing known free fetal nucleic acid concentrations, this embodiment uses a male fetal control sample in which the concentration of free fetal nucleic acid is determined according to the Y chromosome, ie through the above male The method of fetal nucleic acid concentration fy is determined.

b. Count the length of the unique alignment sequence contained in all control samples, from 0 bp to Mbp (M indicates the longest length of the nucleic acid), and determine the number of unique alignment sequences that occur at each length. ;

c. using a certain length as a candidate length range, and dividing a plurality of candidate length ranges according to a step size of 1-2 bp, for example, 1 bp, 2 bp, 3 bp, ..., 100 bp, ..., 300 bp, and counting the control samples in each The frequency of unique alignment sequences that occur within the candidate length range;

d. finding a candidate length of the plurality of control samples that have a unique correlation between the frequency of the sequencing sequence and the concentration of the nucleic acid in the control sample within each candidate length range A combination of degrees or ranges determines a correlation coefficient between each of the candidate length ranges and the concentration of the nucleic acid; wherein the correlation coefficient is calculated by correlation, including linear regression, logistic regression, local weighting, and the like.

Wherein, the candidate length range has a span of 1-300 bp, preferably 1-20 bp. The multiple candidate length ranges have a step size of 1-2 bp.

e. Determine, according to the value of the correlation coefficient, at least one candidate length range or a candidate length range combination as the predetermined range.

(2) counting the frequency at which the unique alignment sequence appears within the predetermined range based on the number of unique alignment sequencing sequences whose length falls within a predetermined range;

(3) determining a female fetal nucleic acid concentration fs in the sample based on a predetermined function based on the frequency of the unique aligned sequencing sequence within the predetermined range.

The predetermined function is obtained by the following steps:

a determining, in the plurality of control samples, respectively, a frequency at which the unique alignment sequence is generated within the predetermined range, the predetermined range in the control sample and the frequency of the unique alignment sequencing sequence are determined by the aforementioned predetermined range get;

b. Linearly fitting the frequency of occurrence of the unique alignment sequence insert within the predetermined range within the predetermined range to a known nucleic acid concentration to determine the predetermined function.

Preferably, the predetermined function is d = -0.3215 x p + 1.62562, wherein d represents the free fetal nucleic acid concentration and p represents the frequency at which a unique alignment sequence occurs within the predetermined range.

3. Calculate the ratio of the concentration fm of the microdeletion microrepetition fragment to the male fetal nucleic acid concentration fy rmY=fm/fy, or calculate the ratio of the concentration fm of the fragment containing the microdeletion microrepetition to the female fetal nucleic acid concentration fs rms=fm/ Fs;

4. Calculate rmY≧2 according to the missing copy number or calculate the copy number to obtain rmY≧6, then judge it as untrustworthy and filter out the false positive result;

Alternatively, if the missing copy number is calculated by rms≧2 or the repeated copy number is calculated to obtain rms≧6, it is determined to be untrustworthy, and the false positive result is filtered out;

Filtering false positives is to remove the effects of multiple copies and make the results more accurate.

5. Take the fractional part of the rmY dmY, or the fractional part of the rms dms, to determine whether the dmY or dms is positive, otherwise the result is filtered:

If dmY<0.13 or dmY>0.85, dmY is positive;

Or, if dms<0.15 or dms>0.791, dms is positive;

6. Calculate the sum of the concentration fm containing the microdeletion microrepetition fragment and the male fetal nucleic acid concentration fy as amY=fm+fy, or calculate the sum of the concentration fm containing the microdeletion microrepeat fragment and the female fetal nucleic acid concentration fs as ams=fm +fs;

7. If amY is between 0.95-1.05, the microdeletion microrepetitive fragment is considered to be from a mother, and the microdeletion microrepetitive fragment is filtered;

Alternatively, if the ams is between 0.93-1.06, the microdeletion microrepetitive fragment is considered to be from the mother, the microdeletion microrepetitive fragment is filtered, and the fetal chromosomal microdeletion microrepetitive fragment is obtained after filtration.

Example 2

1. Sample collection and processing

One batch of 100 samples was selected, and 2 ml of peripheral blood was extracted for plasma separation.

2, library construction

Library construction can be performed with reference to plasma library construction requirements well known to those skilled in the art.

3. Sequencing

The sequencing process can be performed on a computer basis with reference to sequencing procedures well known to those skilled in the art.

4, data analysis

The sequencing results were obtained by double-end sequencing, and the results of the initial micro-deletion micro-repetition were obtained by the following analysis, and the steps are as follows:

4.1 Alignment, the sequencing results are aligned to the reference genome to determine the location of the unique alignment sequence.

4.2 The reference genome was divided into multiple primary windows according to the length of 20 kb. The number of unique alignment sequences and GC content in each primary window was counted, and the number of sequences falling into the primary window by local weighted regression was used for GC correction.

4.3 For all samples in the batch, perform baseline corrections and inter-batch adjustments for each primary window.

4.4 Combine adjacent primary windows in units of 100, and combine to obtain a plurality of secondary windows, the secondary window having a length of 2M;

4.5 Calculate the T1 value of each secondary window by using the Z test, and filter out the secondary window with the T1 value between -3-3;

4.6 Run the run test on the filtered secondary window to calculate the T2 value, and merge the adjacent two secondary windows with the T2 value between -3-3 into the final window according to the T2 value;

4.7 Repeat steps 4.5-4.6 until they cannot be merged;

4.8 According to the Z test, the final window obtained by the final combination was calculated, and the micro-deletion micro-repetition results were calculated. A total of 19 samples with micro-deletion micro-duplication were detected.

Table 1 Results of 19 samples

Table 1. Table shows the results of 19 samples, the first column is the id of the sample, the second column is the microdeletion microrepetition, and the third column is the microdeletion microrepetition length of the chromosome, fourth The column is the detected T value.

4.9 Calculate the concentration of micro-deletion micro-repeat fragments based on the results of micro-deletion micro-repeats. The specific steps are as follows:

Calculating an average depth d1 of the primary window containing microdeletion microrepetitions in each sample;

Calculating the average depth d2 of the primary window without microdeletion microrepetitions;

Calculating the concentration fm of the microdeletion microrepetitive fragment;

Calculate the fetal nucleic acid concentration. The table for the above 19 results is as follows:

Table 2. Fetal nucleic acid concentration information for 19 samples

4.10 Calculate the fetal concentration according to the ratio of chrY, and obtain the male fetal concentration of 8 samples. The specific steps are as follows:

Removing the primary window in which the number of unique alignment sequences adjusted by GC modification in the chromosome chrY is more than 5 times the number of average sequences;

Calculating the average depth dy of the primary window in chrY;

Calculate the male fetal nucleic acid concentration fy, the results are as follows:

Table 3. Results of fetal nucleic acid concentrations estimated from chrY in 19 samples

4.11 Calculate the concentration of the fetus according to the length of the fragment, and obtain the female fetus concentration of 11 samples. The specific steps are as follows:

A total of 41 male samples were counted in the whole batch to find the region with high frequency and fetal concentration. The selected region was 179bp-206bp, and the correlation coefficient was R=-0.9056996.

The relationship between the frequency of the nucleic acid in the remaining 11 samples ranging from 179 bp to 206 bp in the range of 179 bp to 206 bp was calculated as a function of the free fetal nucleic acid concentration. The selected region was linearly fitted with 179 bp to 206 bp to obtain the relationship d. = a × p + b, where d represents the concentration and p represents the frequency of occurrence, and a and b are calculated to be -0.3215 and 1.62562, respectively.

The results of the female fetus samples were calculated according to the fitting, and the results were as follows:

Table 4. Fetal nucleic acid concentrations calculated from 19 samples based on fragment length

4.12 Screen for microdeletion microrepetition results.

For male fetuses:

Calculating the ratio of the concentration fm of the microdeletion microrepetitive fragment to the male fetal nucleic acid concentration fy rmY=fm/fy;

Filtration is performed according to the copy number, and the missing fragment having the copy number rmY value of 2 or more is filtered, and the fragment having the repeated copy number rmY value of 6 or more is filtered.

Take the fractional part of rmY to get dmY.

For the remaining fragments, fragments having a dmY greater than 0.13 and less than 0.85 were filtered.

Calculating the sum of the concentration fm containing the microdeletion microrepeat fragment and the male fetal nucleic acid concentration fy is amY=fm+fy;

Fragments with amY > 0.95 and amY < 1.05 were filtered to obtain samples of male fetuses containing microdeletions and microduplications.

For female fetuses:

Calculating the ratio of the concentration fm of the microdeletion microrepetition fragment to the female fetal nucleic acid concentration fs rms=fm/fs;

Filtering according to the copy number, filtering the missing fragments with the copy number rms value of 2 or more, and filtering the fragments with the repeated copy number rms value of 6 or more.

Take the fractional part of rms to get dms.

For the remaining fragments, fragments with dms greater than 0.15 and less than 0.791 were filtered.

Calculating the sum of the concentration fm containing the microdeletion microrepeat fragment and the female fetal nucleic acid concentration fs is ams=fm+fs;

Fragments with ams > 0.93 and ams < 1.06 were filtered to obtain a sample of female fetuses containing microdeletions and microduplications.

The results obtained positive are shown in Table 5 below:

Table 5. Microdeletion microrepetition results after filtration

sample 8 chromosome starting point 19394465 End position 27194537 Microdeletion microrepetition length 7.80M T value 5.030 Microdeletion microrepetition calculated concentration 0.119863 Fetal concentration calculated according to chrY Fetal concentration calculated from the length of the segment 0.126509

The results of the micro-deletion micro-replication can be filtered to obtain a large number of false positives, and an accurate result is obtained, see FIG. In the figure, the abscissa indicates the number of the sample, and the ordinate indicates the concentration, where fm indicates the concentration estimated by the microdeletion microrepetition, fy indicates the concentration estimated by the male fetus sample according to chrY, and fs indicates the concentration estimated by the fetus according to the fragment. It can be seen that, after judging by the above criteria, the sample numbered 28 is a fetal sample that ultimately contains micro-repetition results.

Implementation 3

The method for determining the microdeletion microrepetition in the fetal chromosome in this embodiment is the same as that in the second embodiment. Again, the difference is that in step 4.2, the 40 kb window is divided.

Implementation 4

The method for determining the microdeletion microrepetition in the fetal chromosome in this embodiment is the same as that in the second embodiment, except that the step 4.4 is performed in units of 200, and the length of the secondary window obtained after the combination is 4M.

Implementation 5

The method for determining microdeletion microduplication in fetal chromosomes in this example is the same as in Example 2, except that 40 male samples are used in step 4.11, the selected region is 185-204 bp, and the correlation coefficient is R=-0.87.

Using the selected region 185-204bp for linear fitting, the relationship d = a × p + b is obtained. In the formula, d represents the concentration, and p represents the frequency of occurrence. Calculated a and b are respectively 0.0334 and 1.6657.

The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to be limiting, and the present invention will be described in detail with reference to the preferred embodiments of the present invention. Neither should the spirit and scope of the technical solutions of the present invention be deviated.

Claims (13)

A method for determining microdeletion microrepetitions in a fetal chromosome, characterized by comprising the steps of: S1, obtaining a concentration fm containing a microdeletion microrepeat fragment; S2, obtaining a male fetal nucleic acid concentration fy or a female fetal nucleic acid concentration fs; S3. Calculating a ratio rmY=fm/fy of the concentration fm containing the microdeletion microrepeat fragment to the male fetal nucleic acid concentration fy, or calculating a ratio of the concentration fm containing the microdeletion microrepeat fragment to the female fetal nucleic acid concentration fs rms=fm/fs ; S4, calculating rmY or rms according to the missing copy number or the repeated copy number, filtering out the false positive; S5, taking the fractional part dmY of rmY, or the fractional part dms of rms, determining whether dmY or dms is positive, otherwise filtering out the result; S6. Calculating the sum of the concentration fm containing the microdeletion microrepeat fragment and the male fetal nucleic acid concentration fy is amY=fm+fy, or calculating the sum of the concentration fm containing the microdeletion microrepeat fragment and the female fetal nucleic acid concentration fs is ams=fm +fs; S7, filtering the micro-deletion micro-repeat fragments according to the judgment principle, and filtering to obtain a fetal chromosome micro-deletion micro-repeat fragment. The method according to claim 1, wherein the concentration fm of the microdeletion microrepetitive fragment in the step S1 is obtained by the following steps: S11. Obtaining a primary window containing no microdeletion microrepetition according to a primary window containing microdeletion microrepetitions, calculating a total number of primary windows containing microdeletion microrepetitions and a total number of primary windows containing microdeletion microrepetitions, and The total number of sequences of primary windows that do not contain microdeletion microduplications and the total number of primary windows that do not contain microdeletion microduplications; S12. Obtain an average depth d1 of the primary window containing the microdeletion microrepetition, d1= Total number of primary windows containing microdeletion microrepetitions / number of primary windows containing microdeletion microduplications; S13, obtaining an average depth d2 of the primary window containing no microdeletion microrepetition, d2 = total number of primary windows without microdeletion microrepetitions / total number of primary windows without microdeletion microrepetitions; S14. Calculating a concentration fm containing microdeletion microrepeats, fm=2×-d2-d1-/d2. The method of claim 2 wherein said final window containing microdeletion microduplication is obtained by the following steps: S111. Perform nucleic acid sequencing on a biological sample containing free nucleic acid to obtain a sequencing result composed of a plurality of sequencing data; S112, aligning the sequencing result with a reference genome to construct a unique alignment sequencing sequence set, each of the unique alignment sequencing sequence sets can only match one position of the reference genome; S113. Determine a length of each unique alignment sequencing sequence in the unique alignment sequencing sequence set; S114, dividing the reference genome into a plurality of primary windows according to a predetermined length, the predetermined length being 1 bp-5M; S115. Count the number of each unique alignment sequence that falls into each primary window; S116: performing GC correction on the number of sequences falling in the primary window, and performing batch-to-batch adjustment on the corrected result; S117. Combine a predetermined number of adjacent primary windows into a plurality of secondary windows, and determine a number of sequences in each secondary window. S118, performing statistical tests on each secondary window, and calculating a T1 value, according to the The T1 value filters the secondary window; S119. Perform a statistical test on the filtered secondary window, calculate a T2 value, and merge two adjacent secondary windows having no significant difference into an ultimate window according to the T2 value; S120, repeat steps S118-S120 until they cannot be merged; S121: Perform a hypothesis test on the final window obtained by the final combination, and obtain an ultimate window containing the micro-deletion micro-repeat. The method according to claim 3, wherein the predetermined length in the step S114 is 1 bp to 5 M, and the predetermined number in the step S117 is 5 to 100. The method according to claim 3, wherein the value of T1 in the step S118 comprises calculating according to a Z test or a T test, and the filtering is filtering out the secondary window in which the T1 value is between -3 and 3. . The method according to claim 3, wherein the value of T2 in step S119 comprises calculating according to a rank sum test, a symbol test or a run test, wherein the non-significant difference is a T2 value of two adjacent windows. Between -3-3. The method according to claim 3, wherein the hypothesis test in the step S121 comprises calculating according to a Z test or a T test, that is, when the statistic of the test is >3 or <-3, it is determined to contain the microdeletion microrepetition. The ultimate window. The method according to claim 1, wherein said male fetal nucleic acid concentration fy in said step S2 is obtained by the following steps: S211, sequencing a biological sample containing free nucleic acid to obtain a sequencing result composed of a plurality of sequencing data; S212. Determine, according to the sequencing result, the number of unique alignment sequences in the Y chromosome in the sample that fall into the primary window; S213. Counting the number of unique alignment sequences in each primary window on the Y chromosome The sum and the total number of primary windows; S214, obtaining an average depth dy of the primary window in the Y chromosome, dy=the sum of the number of unique aligned sequencing sequences on the Y chromosome/the number of primary windows on the Y chromosome; S215, obtaining a male fetal nucleic acid concentration fy, fy=2×dy/d2, the d2 is the average depth of the primary window without the microdeletion microrepetition, and d2=the total number of primary windows without the microdeletion microrepetition/ The number of primary windows that do not contain microdeletion microduplication. The method according to claim 1, wherein the female fetal nucleic acid concentration fs in the step S2 is obtained by the following steps: S221, sequencing a biological sample containing free nucleic acid to obtain a sequencing result composed of a plurality of sequencing data; S222. Determine, according to the sequencing result, a number of unique alignment sequencing sequences whose length falls within a predetermined range in the sample; S223. Determine a frequency of occurrence of a unique alignment sequencing sequence within the predetermined range based on the number of unique alignment sequencing sequences whose length falls within a predetermined range; S224. Determine a female fetal nucleic acid concentration fs in the sample according to a predetermined function according to a frequency at which the unique alignment sequence appears within the predetermined range. The method according to claim 1, wherein the step S4 further comprises: if the rmY ≧ 2 is calculated according to the missing copy number or the repeated copy number is calculated to obtain rmY ≧ 6, the determination is not Letter, filter out false positive results; Alternatively, if rms≧2 is calculated based on the missing copy number or the repeated copy number is calculated to obtain rms≧6, it is determined to be unreliable, and the false positive result is filtered out. The method according to claim 1, wherein the step S5 further comprises: if dmY<0.13 or dmY>0.85, the dmY is positive; Alternatively, if dms < 0.15 or dms > 0.791, dms is positive. The method according to claim 1, wherein the determining principle in the step S7 is: if the amY is between 0.95 and 1.05, the fragment of the microdeletion microrepetition is considered to be from the mother, and the microdeletion is filtered. Micro-repetitive fragment; Alternatively, if ams is between 0.93-1.06, the microdeletion microrepetitive fragment is considered to be from the mother, and the microdeletion microrepetitive fragment is filtered. An apparatus for determining microdeletion microrepetitions in a fetal chromosome, characterized by comprising: a micro-deletion micro-repeat fragment concentration calculating device for obtaining a concentration fm containing a micro-deletion micro-repeat fragment; a fetal nucleic acid concentration obtaining device for obtaining a male fetal nucleic acid concentration fy or a female fetal nucleic acid concentration fs; a ratio calculating device for calculating a ratio rmY=fm/fy of a concentration fm containing a microdeletion microrepetition fragment to a male fetal nucleic acid concentration fy, or calculating a ratio rms of a concentration fm containing a microdeletion microrepeat fragment to a female fetal nucleic acid concentration fs =fm/fs; a first filtering device for calculating rmY or rms according to the missing copy number or repeated copy number, filtering out false positives; a second filtering device, configured to take the fractional part dmY of rmY, or the fractional part dms of rms, to determine whether dmY or dms is positive, or filter out the result; And a value calculation device for calculating the sum of the concentration fm containing the microdeletion microrepetition fragment and the male fetal nucleic acid concentration fy as amY=fm+fy, or calculating the concentration fm containing the microdeletion microrepeat fragment and the female fetal nucleic acid concentration fs And ams=fm+fs; The third filtering device is configured to filter the micro-deletion micro-repeat fragments according to the determination principle, and filter to obtain a fetal chromosome micro-deletion micro-repeat fragment.

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