WO2016049877A1 - Detecting methods and systems based on str typing technology for non-invasive prenatal testing - Google Patents

Abstract

The present invention provides a detecting method and system for non-invasive prenatal testing. The method comprises the following steps: 1) selecting STRs and designing primers; 2) amplifying nuclear acids obtained from the alleged father and the pregnant mother by said primers to obtain amplified sequence; 3) sequencing said amplified sequence to obtain sequencing sequences; 4) obtaining corresponding base sequences for each STR with regard to said above sequences, and making STR typing on the basis of said base sequences; 5) screening obtained STR genotypes; and 6) calculating related parameters of paternity test.

Description

Detection method and system based on STR typing technology in non-invasive prenatal identification Technical field

The invention relates to the field of paternity testing, in particular to a detection method and system for non-invasive prenatal paternity testing.

Background technique

Paternity testing is a technique that uses forensic science, biology, and genetics, especially using genetic fingerprinting to determine whether two individuals have parental genetic relationships [1-3]. The existing paternity testing techniques mainly use polymerase chain reaction (PCR), restricted fragment length polymorphisms (RFLP) and short tandem repeat polymorphisms. Techniques are used to calculate the paternity index and the probability of parentality to make judgments [4].

In recent years, with the need for prenatal paternity testing in judicial cases, especially rape cases, paternity testing techniques have gradually been applied in this field. Existing methods are based primarily on invasive sampling, including chorionic villus sampling (CVS) and amniocentesis, but such sampling can pose a certain risk of miscarriage. Amniocentesis may have a risk of miscarriage from 1/300 to 1/500 [5] and may cause some other complications [6-7]. Chorionic puncture also has a similar risk of miscarriage, and 1 in 3000 causes fetal limb hypoplasia, especially when the sampling time is 10 weeks ago [8-9].

The discovery of fetal cell-free DNA (cff DNA) in maternal plasma in 1997 made non-invasive prenatal testing possible, and non-invasive prenatal testing would avoid the risk of invasive sampling [10]. Although cff DNA was discovered in 1997, reliable non-invasive prenatal testing based on maternal peripheral blood has not been successfully developed because cff DNA is low in maternal plasma. It is generally considered to be <20%, and its content is lower in pregnant women with low gestational age, and cff DNA is a highly fragmented sample with a fragment size of about 140-196 bp [12]. How to effectively overcome such barriers and apply them to the field of non-invasive paternity testing is urgently needed.

Based on existing invasive sampling techniques (chorionic puncture or amniocentesis), the sampling time should not be earlier than 10 weeks. In the case of general rape, the induction time is about 10 weeks. Non-invasive prenatal paternity testing technology came into being, but most of the existing technologies are based on the high cost of SNP typing technology [13], while the detection method based on STR typing technology can only detect samples with male fetus and need to be constructed. The library has certain limitations [14].

Therefore, it is urgent to develop a detection procedure based on the existing STR typing method but suitable for all pregnant women, especially low gestational age (6-9 weeks).

Summary of the invention

The present invention provides, for the first time, a detection method or system based on short tandem repeat (STR) typing techniques for use in non-invasive prenatal paternity testing, and the detection method or system of the invention includes existing Sanger-based sequencing, The second-generation high-throughput sequencing technology, the first innovative use of high-throughput database construction strategy, can be quickly and large-scale (for example, up to 144 samples at a time) screening for second-generation sequencing A library of platforms (including but not limited to Illumina Hiseq or Miseq, Ion Torrent). The detection method or system of the invention is not limited to the sex of the fetus, and can be applied to the gestational age of 6 to 24 weeks, and can effectively perform prenatal non-invasive paternity testing.

The invention provides a detection method and system based on STR typing technology in non-invasive prenatal paternity testing, the method and system comprising the following steps:

1) Select STR (for example, according to the STR database (http://www.cstl.nist.gov/strbase/) and related literature reports) and design primers for these STRs;

2) performing nucleic acid amplification from the experimentally relevant sample with the primer pair to obtain an amplified sequence, the experimental related sample including a peripheral blood sample (for example, 5 ml) of the pregnant woman and a sample from the suspected parent, the peripheral blood of the pregnant woman Preferably, the sample is plasma separated by centrifugation after sampling (for example, within 2 hours) to obtain a pregnant woman's a plasma sample and a white blood cell sample, the sample from the suspected father is preferably whole blood, saliva, blood spots, hair;

3) sequencing the amplified sequence to obtain a sequence thereof;

4) For the above sequence, the corresponding base sequence of each STR is obtained, and STR typing is performed according to the base sequence, for example, the base sequence source is optionally a generation Sanger sequencing sequence and a second generation high-throughput sequencing sequence, for Sanger The sequence will be STR-typed by IDX software included in the ABI 3130 sequencer. If D13S317 is available as type 9,11, STR typing will be performed by software for the second-generation sequencing sequence;

5) Screening and screening of the obtained STR types (such as D13S317 type 9, 11 type), the standard is: 1) specific STR loci such as D13S317 type results, 2) specific STR loci such as D13S317 type The result cannot exceed three states. For example, if D13S317 is 9, 11, 12, 14, the site is unqualified and cannot be used for the next calculation;

6) Calculate the parameters related to paternity testing, including CPI and W, where CPI is the cumulative paternity coefficient, which is obtained by multiplying the paternity index of each STR locus, and is judged as a positive in paternity test. An important indicator of patriarchal relationship, when the CPI is greater than 400, the general patriarchal power can be affirmed (http://baike.baidu.com/view/29220.htm?fr=aladdin), W is the parent-child possibility, that is, when W When calculated as 99%, it means that 99% of the suspected father is the father of the fetus, and 1% may not be the father of the fetus.

The detection is not limited to the sex of the fetus, and is suitable for low gestational age detection, and can be detected by this method when the gestational age reaches 6 weeks.

The selecting STR includes:

Sixteen autosomal STR loci were selected, namely CSF1PO, FGA, TH01, TPOX, vWA, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, Penta D, Penta E, D2S1338, Amelogenin,

Sixteen Y chromosome STR loci were selected, namely DYS438, DYS391, DYS392, DYS393, DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385, DYS439, DYS635, Y-GATA-H4, DYS437, DYS448,

Eight X-stained human STR loci were selected, namely DXS7130, DXS7424, DXS101, DXS10147, DXS7132, DXS6803, HPRTB, DXS7423.

The method and system of the present invention make maximum use of the existing STR typing system and the utilization of the second generation sequencing, and effectively perform non-invasive prenatal testing on pregnant women with low gestational age and non-fetal sex.

The invention can also be described as:

A detection method based on STR typing technology in non-invasive prenatal paternity testing, the method comprising the following steps:

1) Select STRs and design primers for these STRs;

2) performing nucleic acid amplification of a sample from a pregnant woman and a suspected father with the primer pair to obtain an amplified sequence;

3) sequencing the amplified sequence to obtain a sequencing sequence;

4) for the above sequencing sequence, obtaining the corresponding base sequence of each STR, and performing STR typing according to the base sequence;

5) Screening and screening of the obtained STR type, the standard is:

The STR locus retaining the type result and the type result not exceeding the three states is used for the next calculation;

6) performing a parent-child identification related parameter calculation and determining a parent-child relationship according to the parameter, wherein the parameter includes CPI, W, wherein the CPI is an accumulated patriarchal coefficient, which is obtained by multiplying the parental weight coefficient PI of each STR site. W is the patriarchal probability, indicating the possibility of parenting,

Preferably, the detection is not limited to fetal sex, and is suitable for low gestational age detection, and can be detected by this method when the gestational age reaches 6 weeks.

2. The method of item 1, wherein the sample from the pregnant woman comprises a peripheral blood sample of the pregnant woman, and the peripheral blood sample of the pregnant woman is preferably subjected to plasma separation by centrifugation after sampling (for example, within 2 hours) to obtain a plasma sample of the pregnant woman and White blood cell sample.

3. The method of item 1 or 2 above, wherein the sample from the suspected father is whole blood, saliva or blood spots.

4. The method of any of the above 1-3, wherein the library construction method suitable for second-generation high-throughput sequencing employs a Wafergen platform construction method based on a large-scale library-based nanopore technique.

5. The method according to any one of the above 1 to 4, wherein the base sequence source is optionally a generation Sanger test Sequence and second-generation high-throughput sequencing sequences, for example, the Sanger sequence will be STR-typed by the IDX software that comes with the ABI 3130 sequencer, such as D13S317 being type 9,11, for example, for the second-generation sequencing sequence STR typing is performed by software.

6. The method of any of the above 1-5, wherein the formula of the paternity index PI is: PI=X/Y,

X = the probability that a man with a suspected father's genetic phenotype is the biological father of the child, and Y = the probability that the random man is the biological father of the child.

7. The method of item 6 above, the patriarchal accumulation index

Where PIi is the paternity index PI when the STR locus is i.

8. The method of item 7 above, the calculation formula of the patriarchal probability W is: W=CPI/(CPI+1).

9. The method of any of the above 1-8, wherein the CPI is greater than 400, which is a positive patriarchy.

10. The method of any of the above 1-9, wherein the detecting is not limited to the sex of the fetus.

11. The method of any of the above 1 to 10, wherein the test is suitable for low gestational age detection; for example, the gestational age is up to 6 weeks for detection.

12. The method of any of the above 1-11, wherein the selecting the STR comprises:

Sixteen autosomal STR loci were selected, namely CSF1PO, FGA, TH01, TPOX, vWA, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, Penta D, Penta E, D2S1338, Amelogenin,

Sixteen Y chromosome STR loci were selected, namely DYS438, DYS391, DYS392, DYS393, DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385, DYS439, DYS635, Y-GATA-H4, DYS437, DYS448,

Eight X-stained human STR loci were selected, namely DXS7130, DXS7424, DXS101, DXS10147, DXS7132, DXS6803, HPRTB, DXS7423.

13. A combination of STR loci comprising:

16 autosomal STR loci, namely CSF1PO, FGA, TH01, TPOX, vWA, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, Penta D, Penta E, D2S1338, Amelogenin,

16 Y chromosome STR loci, namely DYS438, DYS391, DYS392, DYS393, DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385, DYS439, DYS635, Y-GATA-H4, DYS437, DYS448,

Eight X-stained human STR loci were DXS7130, DXS7424, DXS101, DXS10147, DXS7132, DXS6803, HPRTB, DXS7423.

14. An information analysis method for STR analysis of parent-child relationship:

1) Screening of STR loci with type results, and the results of the type not exceeding three states

2) performing the calculation of the relevant parameters of the paternity test, and determining the parent-child relationship according to the parameters,

The parameters include CPI, W, where CPI is an accumulated patriarchal coefficient, which is obtained by multiplying the paternity coefficient PI of each STR locus, and W is a paternity probability, indicating a parent-child probability.

In one embodiment, the formula for calculating the paternity index PI is: PI=X/Y,

Where X = the probability that a man with a suspected father's genetic phenotype is the biological father of the child, and Y = the probability that the random man is the biological father of the child;

Preferably, the patriarchal accumulation index

Where PIi is the paternity index PI when the STR locus type is i;

Preferably, when the CPI is greater than 400, it is a positive patriarchal power;

Preferably, the formula for calculating the patriarchal probability W is: W=CPI/(CPI+1).

DRAWINGS

Figure 1 shows a high-throughput chip.

Figure 2 is a road map of the implementation process of the entire invention.

Figure 2 2100 detection peak map in library quality testing.

detailed description

definition

The "STR" referred to in the present invention is a short tandem repeat sequence, also known as microsatellite DNA, and the repeating unit is 2-6 bp, repeating times 10-60 times, gene fragment, below 400 bp.

The term "STR type" as used in the present invention refers to the STR genotype, that is, the genotype of STR, and the STR has two alleles, such as D13S317 type, which can be 9,11 (http://www.cstl.nist.gov /strbase/glossary.htm). Specifically, in the STR type, different numbers represent different copy numbers of the site, such as D8S1179 type is 12, 14, which means that there is a copy number of 12 core sequences at the gene position of D8S1179, and The position of one allele has a copy number of 14 core sequences.

As used herein, "barcode" refers to a mark consisting of a set of base sequences arranged in a certain order to indicate certain information.

The term "internal primer" as used in the present invention refers to a primer for a specific site to which a specific linker sequence is added, i.e., a specific common linker is added to 5' of the original common primers F and R.

As used herein, "external primers" (including lateral forward primers and reverse primers) refer to primers containing specific public linkers and sequences adapted to the second generation sequencing sequence.

In a specific embodiment, the method and system of the present invention are specifically implemented as follows:

1. STR locus screening and evaluation, primer design and testing; use the kit to understand the screening, the content includes the number of STR loci contained in the kit, the specific conditions of the STR locus, and which locus The length of the primer after amplification, since the length of free DNA in plasma is about 170 bp, the amplification product of the primer is not more than 300 bp in principle;

2. The experimental related samples required for the relevant samples of the experiment include 5 ml of the peripheral blood sample of the pregnant woman, and the plasma is separated by centrifugation within 2 hours after the sampling, and the plasma sample and the white blood cell sample of the pregnant woman are obtained, and the sample from the suspected father is optional. Blood, saliva, blood spots, and corresponding DNA extraction of experimental samples including, but not limited to, plasma, whole blood and blood spot DNA extraction;

3. Quantitative and quality assessment of extracted DNA;

4. Amplify a specific STR locus;

5. Sequencing the amplified products, including Sanger sequencing, Ion Torrent sequencing but Not limited to this;

6. Analyze the sequencing data to obtain a specific STR type;

7. Screening and evaluation of the obtained STR type, and performing parent-child identification related parameters including CPI calculation;

8. Further confirm the evaluation of the results obtained.

A detection method and system based on traditional STR typing technology in non-invasive prenatal paternity testing, the specific implementation steps are as follows:

1. STR locus screening, according to http://www.cstl.nist.gov/strbase/ data for screening, specifically according to the frequency distribution of STR gene, polymorphism combined with literature reports, select commonly used effective STR point. Primer design and testing, the test content includes internal primer specific verification, the specific process is as follows:

The inner primer is usually dry powder and stored at -20 ° C when it is not dissolved. The primer should be centrifuged at 14000 rpm for 5 min before dissolution, and then the primers are dissolved according to the number of nmol given according to the primer order. (The primer is dissolved to 10 μM, and the volume of the solution is added by 100 times the number of nmol, for example, the primer is 2.5 nmol, and 250 μL of water is added. , the primer concentration is 10 μM). Before the primer is dissolved, the ultra-clean workbench needs to be sterilized for 30 minutes in advance. When the solution is dissolved, the wind cannot be turned on. The wind can be disconnected to remove the pollution. When the wind is opened, all the primer tube covers should be covered. After adding the solution, the Votex was mixed and centrifuged. Pipette the appropriate amount of the inner primer solution to a 96-well plate for later use. The inner primers were diluted to 250 nM each (2.5 μL Primer F + 2.5 μL Primer R + 95 μL H ₂ O) in a 96-well plate, sealed with an aluminum seal and then covered with a plastic film. Store at -20 °C when not in use.

Internal primer verification system:

Primer verification program:

95 ° C 5 min;

10 cycles:

95°C 15sec

60 ° C 30 sec

72 ° C 1 min;

2 cycles:

8 loops:

95°C 15sec

60 ° C 30 sec

72 ° C 1 min;

2 cycles:

8 loops:

95°C 15sec

60 ° C 30 sec

72 ° C 1 min;

15 cycles:

The above PCR products were subjected to electrophoresis detection, and the results are shown in Fig. 1, only a part of which is shown. For illustrative purposes only.

2. The kit can be screened, specifically for the content including the number of STR loci contained in the kit, the specific conditions of the STR locus, and the length of the primer after amplification, due to the free in plasma The length of the DNA is about 170 bp, so the amplification product of the primer is not more than 300 bp in principle, and preliminary filtration is performed according to the above criteria, and the selected kit is tested. The experiment included the whole blood DNA of the S1 sample named S1-blood, the S1 whole blood DNA was randomly broken to 200 bp. The interrupted DNA sample was named S1-blood-cut, and the plasma extracted DNA sample of the S1 sample was named S1-Plasma. Amplification was performed using the selected kit, and Sanger sequencing was performed, and STR typing was performed using related software such as IDx, GeneMapper 4.0, and judged based on the consistency of the obtained results. The results are shown in Table 1:

Table 1:

According to the STR database (http://www.cstl.nist.gov/strbase/) and related literature reports, 16 autosomal STR loci, 16 Y chromosome STR loci, and 8 X chromosome STR loci were selected, and Primers designed for use on the wafergen platform (available at http://www.wafergen.com/seq-ready/custom/) are listed in the following table:

MiniFiler^TMPCR Amplification Kit和AmpFLSTR Yfiler kit，但不限于此。In addition to the primers designed by the inventors themselves, other companies have also selected kits for STR amplification, such as

MiniFiler ^TM PCR Amplification Kit and AmpFLSTR Yfiler kit, but are not limited to this.

3. DNA extraction

1) Plasma DNA extraction

DNA extraction was performed according to the SNAVO or Tiangen DP316 extraction kit operating instructions. The sample was collected from 5 ml of peripheral blood of pregnant women and centrifuged within 2 hours to obtain plasma and white blood cell samples. The process is as follows:

Peripheral blood separation steps:

4. Allow to stand at room temperature for half an hour, centrifuge at 1600 g for 10 minutes at 4 ° C. After centrifugation, dispense the supernatant (plasma) into a plurality of 2.0 ml centrifuge tubes, taking care not to absorb the middle during the pipetting process. Layer of white blood cells.

5. Centrifuge the dispensed 2.0 ml centrifuge tube at 16000 g (normal 14000 g) for 10 minutes at 4 ° C to remove residual cells and transfer the supernatant to a new 1.5 ml or 2.0 ml centrifuge tube. Plasma needed. Label it - 80 ° C to save.

Briefly, 600 ul of the separated pregnant women's plasma was extracted, and 3 tubes were aliquoted, and 200 ul of each tube was separately extracted. The volume of the total re-dissolved DNA was 20 ul. If the volume was too large, it could be concentrated by a vacuum concentrator to achieve a suitable volume.

2) Whole blood DNA extraction

DNA extraction was performed according to the extraction instructions for whole blood according to the QIAamp DNA Mini kit or the DNeasy 96 Blood & Tissue Kit extraction kit instructions. The sample was obtained from the white blood cells of the pregnant women separated from the previous step and the 5 ml peripheral blood samples collected from the suspected father. In short, the initial volume of the whole blood was extracted to 200 ul, and 100 ul was dissolved.

3) Blood spot DNA extraction

DNA extraction was performed according to the instructions for the extraction of blood spots in the Tiagen DP316 kit. The sample source was taken from a suspected paternal blood spot sample, which in this example was a whole blood sample with a reconstituted volume of 20 ul.

6. DNA quantification

DNA quantification was performed using a POLARstar Omega fully automated microplate reader from BMG, Germany. Quantitative samples included extracted maternal plasma DNA, maternal white blood cell DNA, and suspected paternal whole blood DNA, each requiring 4 ul of quantitation.

7. Amplify specific STR loci

The extracted STR amplification system developed by the inventor's self-developed STR amplification system is all mini-STR, including 16 autosomal mimi-STR, 16 Y chromosome mini-STR and 8 X chromosome mini-STR. The The mini-STR-like amplification primers contain sequences bound by sequencing primers, which can be directly quantified after two rounds of amplification.

A total of 36 sites were selected from 16 autosomal STRs, 16 Y chromosomes, and 4 X chromosomes of 8 chromosomes in self-designed primers. Amplification of the fragment of interest is carried out by high throughput PCR. The high-throughput PCR chip used in the present invention (provided by wafergen, the ordering site is http://www.wafergen.com/seq-ready/custom/ a total of 5184 (36*144) micro PCR reaction wells (Fig. 1 ), each reaction well can carry out a PCR reaction of 100 nL system, and only a total of 30 ng of genomic DNA is required for detecting 54 sites per sample. A round of PCR reaction is carried out by using an external primer containing a barcode and an internal primer designed by the present invention. The target fragment is added, and each sample is added with a barcode and a sequencing adaptor, so that the PCR product does not have to undergo the sequencing process of the sequencing library, and the second-generation high-throughput sequencing is directly performed. The high-throughput PCR amplification chip used in the present invention. Each sample is tested for 36 STR sites, and 144 samples can be PCR-reacted at a time, which is equivalent to constructing 144 second-generation sequencing libraries at one time, which greatly saves the time and cost of sequencing library construction. High-throughput PCR chip support The reagents and the external primers are used for amplification and establishment of the target fragment, and the specific process is as follows:

1) Prepare PCR Mix:

According to the mode of high-throughput chip 36*144, the wafergen platform provides different modes of reaction. The specific situation is as follows. The PCR Mix is prepared according to the following table system. After shaking and mixing, put it on the ice box for backup:

2) Prepare the sample plate:

The concentration of 144 samples to be tested was all diluted to 10 ng/μL, and 20 μL per well was dispensed into two 96-well PCR plates, and PCR Mix, template DNA, and lateral reverse primer were respectively used according to the chip 36×144 mode. According to the 36*144 mode sample loading table provided by wafergen, it is added to the corresponding 96 holes of the 384-well plate. The Outer Reverse Primer is connected with a barcode. Pay attention to the sample according to the pre-arranged barcode number. The specific volume is as follows. Show:

3) Prepare the primer plate:

The primer dry powder was diluted to 100 μM to prepare a mother liquor, and the mother liquor was diluted to 0.25 μM, 100 μL per well was dispensed into a 96-well plate, and PCR Mix was separately used according to the chip 36×144 mode, according to 36* provided by wafergen. Primer pairs in the 144-mode primer-loading table were added to the corresponding 36 wells of the 384-well plate. The specific addition volume is shown in the following table:

Reagent V/well

PCR Mix 12.2μL

Internal primer Pair (0.25μM) 8.1μL

4) On the machine to spot:

Place the sample plate and chip into the corresponding positions of the spotter, select the 36×144 mode, 50nL volume, and start to spot. After the end of the sample for about 30 minutes, the chip was removed, and the chip was sealed with a specific sealing film and centrifuged. Remove the sample plate and seal the sealing film into a -20 ° C freezer for later use. Then put the primer plate and the centrifuged chip into the corresponding position of the spotter, select the mode of 36×144, and the volume of 50nL, and start to spot. After about 30 minutes of sampling, the chip was removed, the parafilm was sealed, centrifuged, and ready for PCR amplification. Remove the primer plate and seal the sealing film into a -20 °C freezer for later use.

5) PCR instrument amplification

Centrifuged the chip, the specific PCR machine runs the following program:

95 ° C 5 min;

10 cycles:

95°C 15sec

60 ° C 30 sec

72 ° C 1 min;

2 cycles:

8 loops:

95°C 15sec

60 ° C 30 sec

72 ° C 1 min;

2 cycles:

8 loops:

95°C 15sec

60 ° C 30 sec

72 ° C 1 min;

15 cycles:

6) Product purification

The amplification product obtained in the above step was centrifuged in a plate centrifuge and collected into a 500 ul EP tube, and 50 ul of 1.5 volumes of magnetic beads was added to carry out product purification.

7) Quality inspection

The library quality was detected by Agilent 2100 Bioanalyzer and real-time PCR (QPCR). The 2100 detection peak map is shown in Figure 3. The fragment range is 200-300 bp, which is consistent with the size of the amplified fragment. The specific detection results are as follows:

The rest of the library quality control requirements are the same as those required for the PGM library.

When the number of samples is small, a second-generation sequencing library can be manually constructed by multi-PCR. In a specific example, the specific process is as follows:

8. Dilute 16 pairs of autosomal STR primers, 16 pairs of Y chromosome STR primers and 8 pairs of X chromosome STR primers to 100 uM. Each 1 ul of the primer dilution was taken into a new 1.5 ml Epp tube, and a total of 80 ul of the primer mixture was obtained, wherein the concentration of each pair of primers was 2.5 uM.

9. Perform the first round of PCR for the purpose of amplifying the target band. PCR is prepared as follows:

The PCR reaction procedure is as follows:

95 ° C 3 min;

30 cycles:

10. Perform two rounds of PCR for the purpose of adding the linker and barcode required for sequencing, and diluting the PCR product of the previous step by 100 times. The Mix prepared by PCR reaction is as follows:

The PCR reaction procedure is as follows:

95 ° C 3 min;

30 cycles:

11. Purification of the above PCR product

The magnetic beads were purified by 1 time.

12. Quality inspection of purified products

13. Agilent 2100 Bioanalyzer and real-time PCR (QPCR) were used for library quality detection. The 2100 detection peak map is shown in Figure 2. The fragment range is 200-300 bp, which is consistent with the size of the amplified fragment. The specific detection results are as follows:

14. Sequencing the amplified product to obtain the STR sequence

Different sequencing methods were performed according to the different amplification methods for obtaining the STR site product of interest in the fourth step.

1) Sanger sequencing

MiniFiler^TMPCR Amplification Kit和AmpFLSTR Yfi ler kit直接扩增的产物用ABI 3130或ABI3730进行测序，通过荧光信号获得指定STR的序列。Take the STR kit commonly used in the market

And MiniFiler ^TM PCR Amplification Kit AmpFLSTR Yfi ler kit amplified product directly sequenced using ABI 3130 or ABI3730, obtain a sequence specified by the STR fluorescent signal.

2) Second generation sequencing platform

Sequencing of the constructed library includes, but is not limited to, the Life Ion Torrent platform, now taking the Ion Torrent platform as an example. The type of sequencing selected was PGM 400. The amount of sequencing data per site per sample was 1000 reads.

7. STR typing of STR sequences

1) Analysis of Sanger data

STR typing was performed according to ABI 3130 software GeneMap 4.0 or id-x and STR database to obtain genotypes of different STR loci in different samples, which were used to calculate the patriarchal coefficient and parent-child index in the next step.

2) Analysis of data from the second generation sequencing platform

The preferred sequencing type in the present invention is Ion Torrent sequencing, so the process is to analyze the data of the Ion Torrent platform, and after the T-map comparison of the offline data, the filter contains the complete STR sequence read and is compared with the STR database. Determine the STR type to obtain the genotypes of different STR loci in different samples, and use it to calculate the patriarchal coefficient and the parent-child index in the next step.

8. Screening for qualified STR loci

MiniFiler^TMPCR Ampl ification Kit和AmpFLSTR Yfiler kit试剂盒，试剂盒分别共包含9个常染色体STR位点和16个Y染色体位点，根据ABI软件id-x的分析结果进行整理汇总。A site with a specific amplified successful STR site and a mother STR type different from the fetus was selected as a follow-up analysis. The results are shown in Table 2. This embodiment uses

MiniFiler ^TM PCR Amplification Kit and AmpFLSTR Yfiler kit kit, the kit contains 9 autosomal STR loci and 16 Y chromosome loci, respectively, according to the analysis results of ABI software id-x.

Table 2 shows the results of autosomal STR typing.

Table 2 shows the results of Y-stained human STR typing: 16 autosomal STR type information, different numbers represent different copy numbers of the site, such as D8S1179 type is 12, 14, which means a gene position in D8S1179 There is a copy number of 12 core sequences, and the position of the other allele has a copy number of 14 core sequences.

Table 2:

Table 3: 16 Y chromosome STR types

Locus mother father Pregnancy plasma DYS456 no 16 16 DYS389 I no 13 13 DYS390 no twenty three no DYS389 II no 30 no DYS458 no 17 17 DYS19 no 15 no DYS385 no 13/17 no DYS393 no 14 14 DYS391 no 10 10 DYS439 no 10 no DYS635 no 20 no DYS392 no 13 no GATA_H4 no 10 10 DYS437 no 14 no DYS438 no 11 no DYS448 no 18 18

The STR typing results are organized into tabular forms according to different loci so that the typing results can be clearly displayed. The specific selection process is:

1. Sort the STR typing results of maternal plasma, pregnant white blood cell DNA, and suspected parent DNA, and draw the table according to Table 2 and Table 3;

2. Analyze whether the STR typing results are accurate and reliable. For the whole genome STR locus, the typing results are generally heterozygous or homozygous, such as the 3-state results, such as D13S317 is 9,11,12 is considered to have If it is polluted, remove this point. There are several cases in which the STR locus of plasma DNA is classified as follows:

The valid information obtained according to the above exhaustive method can be further organized into the following table:

All of the above results were established in the process of amplification of specific STR loci without error. We performed 2 parallel experiments in each experiment and evaluated the accuracy of STR judgment in the early stage. The results showed high accuracy.

9. Calculate the non-parent exclusion rate, the paternity index, the cumulative paternity index, and the paternity coefficient according to the calculation formula.

The non-parent exclusion rate is used to calculate the ability of any such male to be non-parent in the system (such as the last selection of 8 STR loci for non-invasive paternity testing in this example). If the EPP is equal to 99.9%, it means 99.9. The ability of % can exclude non-parents.

The patriarchal index is the ability of each STR site to affirm the patriarchal power. For example, when the PI value of D13S317 is 2, the probability that the suspected father is the father of the fetus can be determined to be twice that of the random male.

The cumulative patriarchal coefficient is the ability of all STR sites of the system to affirm the patriarchy. For example, the last CPI in this embodiment is 6064, which means that the probability that the suspected father is the father of the fetus is 6064 times that of the random male.

The patriarchal coefficient is a possibility to judge the suspected father and the fetus as a father-son relationship. As in this embodiment, W is equal to 99.98%, which means that the probability that the suspected father and the fetus are fathers and sons is 99.98%.

The calculation formula for the non-parent exclusion rate is:

In the above formula, pi and pj are the frequencies of a certain STR type i and j, such as the frequency of D8S1179 type 12 is 0.131;

The formula for calculating the paternity index PI is: PI=X/Y,

In the above formula, X = the probability that the man with the genetic phenotype of the controlled father is the biological father of the child, and Y = the probability that the random man is the biological father of the child;

Patriarchal accumulation index

In the above formula, PIi is the paternity index PI when the STR locus is i;

The formula for calculating the patriarchal probability W is: W(RCP)=CPI/(CPI+1),

In the above formula, CPI is the previously calculated patriarchal accumulation index, and RCP is the abbreviation of "relative chance of paternity".

The results are shown in Table 4. According to the calculation formula of PI, the probability that the male with the genetic phenotype of the controlled father is the biological father of the child and the probability that the random male is the biological father of the child are calculated, so that the PI values of different STR loci are obtained. And multiply and get the cumulative patriarchal coefficient CPI.

Table 4 shows the STR sites filtered according to the filtration criteria, according to the above formula, at http://www.ehstrafd.org/modules/AFGT/form.cfm and http://www.genebase.com/in/dnaMarkerDetail .php? t=y&d=str&m=DYS458 The address of the relevant STR site related type is referred to.

10. Reliability of the judgment result

The analysis method mainly selects 90 males who are not related to biological significance to make parent-child relationship judgment with the given case of the fetus, compares with the suspected father of the test sample, and the result is completely consistent with the actual, thus further confirming the test sample. The parent-child relationship.

references

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Claims (12)

A detection method based on STR typing technology in non-invasive prenatal paternity testing, the method comprising the following steps: 1) Select STR and design primers for the selected STR; 2) performing nucleic acid amplification of a sample from a pregnant woman and a suspected father with the primer pair to obtain an amplified sequence; 3) sequencing the amplified sequence to obtain a sequencing sequence; 4) for the above sequencing sequence, obtaining the corresponding base sequence of each STR, and performing STR typing according to the base sequence; 5) Screen the obtained STR type, the standard is: The STR locus retaining the type result and the type result not exceeding the three states is used for the next calculation; 6) performing a parent-child identification related parameter calculation and determining a parent-child relationship according to the parameter, wherein the parameter includes CPI, W, wherein the CPI is an accumulated patriarchal coefficient, which is obtained by multiplying the parental weight coefficient PI of each STR site. W is the patriarchal probability, indicating the possibility of parent-child. The method of claim 1, wherein the sample from the pregnant woman comprises a peripheral blood sample of the pregnant woman, and preferably the peripheral blood sample of the pregnant woman is preferably subjected to plasma separation by centrifugation after sampling (for example, within 2 hours) to obtain a plasma sample and a white blood cell sample of the pregnant woman. . The method of claim 1 or 2, wherein the sample from the suspected father is whole blood, saliva or blood spots, hair. The method of any one of claims 1-3, wherein the library construction method for second-generation high-throughput sequencing employs a Wafergen platform construction method based on a large-scale library-based nanopore technique. The method of any one of claims 1 to 4, wherein the base sequence is derived from a first generation Sanger sequencing sequence or a second generation high throughput sequencing sequence, for example, the Sanger sequence will be subjected to STR typing by IDX software included in the ABI 3130 sequencer. For example, D13S317 is available. For type 9, 11 , for example, STR typing will be performed by software for the second generation sequencing sequence. The method of any one of claims 1 to 5, wherein the formula of the paternity index PI is: PI=X/Y, Where X = the probability that a man with a suspected father's genetic phenotype is the biological father of the child, and Y = the probability that the random man is the biological father of the child; Preferably, the patriarchal accumulation index

Where PIi is the paternity index PI when the STR locus type is i; Preferably, when the CPI is greater than 400, it is a positive patriarchal power; Preferably, the formula for calculating the patriarchal probability W is: W=CPI/(CPI+1). The method of any of claims 1-6, said detecting being not limited to fetal sex. The method of any of claims 1-7, wherein said detecting is suitable for low gestational age detection; for example, gestational weeks are up to 6 weeks for detection. The method of any of claims 1-8, wherein the selecting the STR comprises: Sixteen autosomal STR loci were selected, namely CSF1PO, FGA, TH01, TPOX, vWA, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, Penta D, Penta E, D2S1338, Amelogenin, Sixteen Y chromosome STR loci were selected, namely DYS438, DYS391, DYS392, DYS393, DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385, DYS439, DYS635, Y-GATA-H4, DYS437, DYS448, Eight X-stained human STR loci were selected, namely DXS7130, DXS7424, DXS101, DXS10147, DXS7132, DXS6803, HPRTB, DXS7423. A combination of STR loci, including: 16 autosomal STR loci, namely CSF1PO, FGA, TH01, TPOX, vWA, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, Penta D, Penta E, D2S1338, Amelogenin, 16 Y chromosome STR loci, namely DYS438, DYS391, DYS392, DYS393, DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385, DYS439, DYS635, Y-GATA-H4, DYS437, DYS448, Eight X-stained human STR loci were DXS7130, DXS7424, DXS101, DXS10147, DXS7132, DXS6803, HPRTB, DXS7423. An information analysis method for STR analysis of parent-child relationship: 1) Screening of STR loci with type results, and the results of the type not exceeding three states 2) performing the calculation of the relevant parameters of the paternity test, and determining the parent-child relationship according to the parameters, The parameters include CPI, W, where CPI is an accumulated patriarchal coefficient, which is obtained by multiplying the paternity coefficient PI of each STR locus, and W is a paternity probability, indicating a parent-child probability. According to the method of claim 11, the formula of the paternity index PI is: PI=X/Y, Where X = the probability that a man with a suspected father's genetic phenotype is the biological father of the child, and Y = the probability that the random man is the biological father of the child; Preferably, the patriarchal accumulation index

Where PIi is the paternity index PI when the STR locus type is i; Preferably, when the CPI is greater than 400, it is a positive patriarchal power; Preferably, the formula for calculating the patriarchal probability W is: W=CPI/(CPI+1).

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