CN108642173A - A kind of method and kit of Non-invasive detection SLC26A4 gene mutations - Google Patents

Abstract

The present invention relates to a kind of method of Non-invasive detection SLC26A4 gene mutations, kits and preparation method thereof, which is characterized in that including steps are as follows：(1) using subject's plasma dna as template；(2) PCR is carried out to expand in advance；(3) Index PCR amplifications are carried out to pre- amplified production；(4) after carrying out library Quality Control to PCR extension products, 150bp both-end sequencings are carried out in Illumina NextSeq；(5) sequence information of sequencing progress bioinformatic analysis is obtained into genetic mutation data.The invention further relates to related kits.

Description

A method and kit for non-invasive detection of SLC26A4 gene mutation

technical field

The invention relates to the field of gene diagnosis, in particular to a method and a kit for non-invasive detection of SLC26A4 gene mutation.

Background technique

The detection of gene mutation can be used to identify whether the genotype of the sample to be tested is wild type or mutant. It can also be used to detect the genetic status of the individual to be tested. For example, whether it contains gene mutations that cause certain diseases.

Many kinds of tumor detection, detection of fetal genetic status are of great value. At present, the genetic mutation screening for the fetus is mainly based on multiple platform systems such as SNP array, and the genome-wide SNP analysis of the fetus and parents is costly; while the conventional polymerase-based sequencing-by-synthesis is difficult to ensure the accuracy of bases, and the occurrence of The base error caused by the sequence noise, so conventional library capture and high-throughput sequencing cannot solve the variation detection bias caused by amplification and sequencing; while the newer cyclic single molecule amplification and resequencing technology (cSMART) technology is affected by its environment Effect of library efficiency and design of back-to-back primer sites.

The discovery of fetal-derived cell-free DNA in the peripheral blood of pregnant women provides the possibility for non-invasive detection of fetal genetic status. The cfDNA in the peripheral blood of pregnant women is derived from the mother and the fetus, and the fetal free DNA (cffDNA) is derived from the placental trophoblast cells, accounting for about 10-20% of the total cfDNA, and the fragment length is about 140-180bp. It is still a technical difficulty to distinguish cffDNA from mother-derived DNA, especially how to detect the imbalance of alleles caused by cffDNA in maternal blood plasma. At present, the international research on cffDNA mainly uses site-specific PCR, MALDI-TOF mass spectrometry, digital PCR and high-throughput sequencing.

The current mainstream non-invasive prenatal detection methods are all based on fetal variant site detection and haplotype detection: through site detection of cfDNA in maternal blood plasma, and then using relative variant dose (RMD) and relative haplotype dose (RHDO) Analyze the relative content of fetal variant sites and haplotypes in maternal plasma. The paternal portion of the fetal genome was deduced by the presence of SNPs in the father only, and the maternal portion of the fetal genome was derived using SNPs that were homozygous for the father but heterozygous for the mother. Papasavva et al. used this method to analyze maternal blood cfDNA, and successfully analyzed its cffDNA genotype and variation sites. Therefore, the detection of variant sites and haplotypes in cffDNA can effectively improve the diagnostic accuracy of fetal disease-causing genes.

There are only a few reports in the world on the methodological research of non-invasive prenatal genetic diagnosis of hereditary deafness. Due to the existence of multiple Indel variant sites in deafness hotspot variants, such as GJB2 (c.299delAT, c.235delC, c.176_191del16), there are still many problems in the cfDNA detection of different platform systems.

It is necessary to develop a more convenient and reliable technique suitable for non-invasive detection of low copy number gene mutations.

Contents of the invention

Therefore, there are problems and needs in the above-mentioned fields. The inventor has developed a fast and accurate single-molecule amplification technology. On the basis of conventional multiple amplification, UMI (Unique Molecular Identifier) molecular tags are added, and UMI is used to perform next-generation sequencing data. Noise reduction to accurately detect low copy number variants: the specific scheme is as follows:

A method for detecting gene mutations, comprising the following steps:

(1) Using the subject's plasma DNA as a template;

(2) Perform PCR pre-amplification;

(3) Carry out Index PCR secondary amplification to the pre-amplification product;

(4) Perform library quality control on the PCR secondary amplification products, and then perform 150bp paired-end sequencing on Illumina NextSeq;

(5) Remove the joints in the sequenced sequence, and then splice it into a sequence to obtain the original template sequence; compare the original template sequence with the human genome, and calculate the unique template sequence set by comparing the UMI molecular tag sequence of the original template sequence ;Use the unique template sequence to calculate the genome coverage for evaluating the specificity of the library; calculate the ratio of the mutant sequence to the reference sequence to calculate the somatic gene mutation rate in the detection region;

It is characterized by:

The primers used in the pre-amplification are the mixture of multiple pairs of primers, and the multiple pairs of primers are used to specifically amplify different sub-target regions in the region to be tested;

All forward primers contain linker sequence A, UMI molecular tag sequence and sub-target region-specific primer sequence from 5' end to 3' end;

All reverse primers contain linker sequence B, UMI molecular tag sequence and sub-target region-specific primer sequence from 5' end to 3' end;

The adapter sequences A in all primer pairs are identical to each other, the adapter sequences B are identical to each other, and the adapter sequences A and B are not identical;

The region to be tested comprises one or several specific gene sequence regions, and the sub-target region refers to DNA fragments in different regions in a specific gene.

Each pair of primers amplifies a sub-target region in the gene, and the UMI molecular tag sequences contained between the primer pairs are different, which is used to ensure that the UMI molecular tag sequences in the DNA amplification fragments derived from the same sub-target region are the same; and the source The UMI molecular tag sequences between the DNA amplification fragments in different sub-target regions are different from each other;

In the secondary amplification of the Index PCR, the secondary amplification primers are sequenced from the 3' end to the 5' end of the adapter sequence and the universal sequencing adapter sequence, and one side of the primer is between the adapter sequence and the universal sequencing adapter sequence. There is also an Index sequence between them; the 3' ends of the forward primer and the reverse primer of the secondary amplification primer are all or part of the adapter sequence A and the adapter sequence B respectively; the Index sequence is used for Mark different samples, use different Index sequences for different samples, and use the same Index sequence for the secondary amplification primers for the same sample.

The above method is characterized in that: the length of the linker sequence A and the linker sequence A is 8-15bp, and the length of the UMI molecular tag sequence is 5-8bp

The above-mentioned method is characterized in that: before performing said Index PCR amplification, the DNA obtained by PCR pre-amplification is carried out to magnetic beads purification;

Aforesaid method is characterized in that: after carrying out described Index PCR amplification, magnetic bead purification is carried out to the amplified DNA.

The test kit for detecting gene mutation is characterized in that it comprises a plurality of primer pairs for specific pre-amplification of different sub-target regions in the region to be tested;

All forward primers contain linker sequence A, UMI molecular tag sequence and sub-target region-specific primer sequence from 5' end to 3' end;

All reverse primers contain linker sequence B, UMI molecular tag sequence and sub-target region-specific primer sequence from 5' end to 3' end;

The adapter sequences A in all primer pairs are identical to each other, the adapter sequences B are identical to each other, and the adapter sequences A and B are not identical;

The UMI molecular tag sequences in DNA fragments from the same source are the same; while the UMI molecular tag sequences are different between DNA fragments from different sources;

The region to be tested comprises one or several specific gene sequence regions, and the sub-target regions refer to different position regions in a specific gene.

The above-mentioned kit is characterized in that it also includes secondary amplification primers for amplifying the pre-amplification products, and the secondary amplification primers are sequentially from the 3' end to the 5' end of the adapter sequence, Universal sequencing adapter sequence, wherein one side primer also includes a section of Index sequence between the adapter sequence and the universal sequencing adapter sequence; the 3' ends of the forward primer and reverse primer of the secondary amplification primer are respectively the adapter sequence All or part of A and the linker sequence B; the Index sequence is used to mark different samples, different samples use different Index sequences, and the secondary amplification primers for the same sample use the same Index sequence.

The above kit is characterized in that: the length of the linker sequence A and the linker sequence A is 8-15bp, and the length of the UMI molecular tag sequence is 5-7bp.

The preparation method of the kit for detecting gene mutation is characterized in that it includes synthesizing and/or assembling multiple primer pairs with the following sequence characteristics,

All forward primers contain linker sequence A, UMI molecular tag sequence and sub-target region-specific primer sequence from 5' end to 3' end;

All reverse primers contain linker sequence B, UMI molecular tag sequence and sub-target region-specific primer sequence from 5' end to 3' end;

The adapter sequences A in all primer pairs are identical to each other, the adapter sequences B are identical to each other, and the adapter sequences A and B are not identical;

The UMI molecular tag sequences in DNA fragments from the same source are the same; while the UMI molecular tag sequences are different between DNA fragments from different sources;

The plurality of primer pairs are used for specific pre-amplification of different sub-target regions in the region to be tested; the region to be tested includes one or several specific gene sequence regions, and the sub-target region refers to a region in a specific gene different location areas.

The above-mentioned preparation method is characterized in that it also includes synthesizing and/or assembling secondary amplification primers for amplifying the pre-amplification products, and the secondary amplification primers from the 3' end to the 5' end It is an adapter sequence and a universal sequencing adapter sequence in sequence, wherein one side primer also includes a section of Index sequence between the adapter sequence and the universal sequencing adapter sequence; the 3' ends of the forward primer and the reverse primer of the secondary amplification primer are respectively It is all or part of the linker sequence A and the linker sequence B; the Index sequence is used to mark different samples, different samples use different Index sequences, and the secondary amplification primers for the same sample use the same Index sequence .

The above preparation method is characterized in that: the length of the linker sequence A and the linker sequence A is 8-15 bp, and the length of the UMI molecular tag sequence is 5-7 bp.

The application of the above method in the detection of GJB2 gene mutation and SLC26A4 gene mutation derives the following technical solutions

A method for non-invasive detection of GJB2 and/or SLC26A4 gene mutations, comprising the following steps:

(1) Using the subject's plasma DNA as a template;

(2) Perform PCR pre-amplification;

(3) Carry out Index PCR secondary amplification to the pre-amplification product;

(4) Perform library quality control on the PCR secondary amplification products, and then perform 150bp paired-end sequencing on Illumina NextSeq;

(5) Remove the joints in the sequenced sequence, and then splice it into a sequence to obtain the original template sequence, compare the original template sequence with the human genome, and calculate the unique template sequence set by comparing the UMI molecular tag sequence of the original template sequence ;Use the unique template sequence to calculate the genome coverage for evaluating the specificity of the library; calculate the ratio of the mutant sequence to the reference sequence to calculate the somatic gene mutation rate in the detection region;

It is characterized by:

In the pre-amplification, a mixture of 26 pairs of primers composed of primers shown in Seq ID No.1-46, 89-91, and 94-96 is used to specifically amplify different sub-target regions of the GJB2 gene;

In the pre-amplification, a mixture of 23 pairs of primers composed of primers shown in Seq ID No.47-88, 92-93, and 97-98 was used to specifically amplify different sub-target regions of the SLC26A4 gene;

Alternatively, a mixture of 49 pairs of primers composed of primers shown in Seq ID No.1-98 is used in the pre-amplification to specifically amplify different sub-target regions of the GJB2 gene and the SLC26A4 gene at the same time; thereby simultaneously detecting the GJB2 gene and SLC26A4 genetic variation;

The region to be tested comprises one or several specific gene sequence regions, and the sub-target region refers to DNA fragments in different regions in a specific gene;

Each pair of primers amplifies a sub-target region in the gene, and the UMI molecular tag sequences contained between the primer pairs are different, which is used to ensure that the UMI molecular tag sequences in the DNA amplification fragments derived from the same sub-target region are the same; and the source The UMI molecular tag sequences between the DNA amplification fragments in different sub-target regions are different from each other;

In the secondary amplification of the Index PCR, the forward and reverse primers shown in Seq ID No.99 and 100 were used.

The above-mentioned method is characterized in that: before performing said Index PCR amplification, the DNA obtained by PCR pre-amplification is carried out to magnetic beads purification;

Aforesaid method is characterized in that: after carrying out described Index PCR amplification, magnetic bead purification is carried out to the amplified DNA.

A kit for non-invasive detection of GJB2 and/or SLC26A4 gene mutation, characterized in that,

A mixture of 26 pairs of primers comprising primers shown in Seq ID No.1-46, 89-91, and 94-96, for specific amplification of different sub-target regions of the GJB2 gene;

A mixture of 23 pairs of primers comprising primers shown in Seq ID No.47-88, 92-93, and 97-98 is used to specifically amplify different sub-target regions of the SLC26A4 gene;

Alternatively, a mixture of 49 pairs of primers comprising primers shown in Seq ID No.1-98 is used to simultaneously specifically amplify different sub-target regions of the GJB2 gene and the SLC26A4 gene; thereby simultaneously detecting variations of the GJB2GJB2 gene and the SLC26A4 gene.

The above kit is characterized in that it also includes secondary amplification primers for amplifying the pre-amplification product, and the forward and reverse primers of the secondary amplification primers are as shown in Seq ID No.99 and 100 Show.

The preparation method of the kit for detecting GJB2 gene mutation is characterized in that it includes synthesizing and/or assembling primers having the nucleotide sequences shown in Seq ID Nos. 1-46, 89-91, 94-96.

Or the preparation method of the kit for detecting SLC26A4 gene mutation is characterized in that it includes synthesizing and/or assembling primers having the nucleotide sequences shown in Seq ID No.47-88, 92-93, and 97-98.

Or the preparation method of the kit for detecting GJB2 gene and SLC26A4 gene mutation, which is characterized in that it includes synthesizing and/or assembling primers having the nucleotide sequences shown in Seq ID No.1-98.

The above-mentioned preparation method is characterized in that it also includes synthesizing and/or assembling secondary amplification primers for amplifying the pre-amplified product, and the forward and reverse primers of the secondary amplification primers are such as Seq ID No. .99 and 100 are shown.

According to the gene mutation detection method provided by the present invention, for the specific gene to be tested, those skilled in the art can design, screen and amplify specific primer sequences for different sub-target regions according to the selected sub-target regions, so that these primer pairs In the same reaction, mutual interference can be minimized to ensure detection accuracy and sensitivity. Then design or select adapter sequences suitable for all primer pairs according to the general design rules of primers, and design UMI molecular tag sequences for different primer pairs, so that primers that amplify DNA fragments from the same source use the same UMI molecular tag sequence, but amplify different primers. The UMI molecules between the primers of the source DNA fragments can be used to obtain the pre-amplification primer set for the specific gene to be tested. It can be used to detect the genetic status of the fetus by cfDNA in the peripheral blood of pregnant women, such as single gene diseases such as deafness genes, and cancer detection by free DNA genes of tumor genes.

In the gene mutation method of the present invention, by introducing UMI molecular tags, the amplified sequencing products with the same UMI sequence are combined through biological information analysis, which greatly reduces the DNA polymerase amplification process and the next-generation sequencing process. Error and false positive rates, with noise reduction enabling accurate detection of low copy number variants.

Experimental example The reliability of the method was verified by detecting the known mutation sites of GJB2 and SLC26A4.

Description of drawings

Fig. 1 is a schematic diagram of the principle of the present invention:

Firstly, the plasma DNA is pre-amplified with mixed primers to amplify the desired site product;

Then the pre-amplification product DNA is purified, the purpose is to remove enzymes and ions in the system;

Secondary amplification of Index PCR was performed on the pre-amplification product to make a library that can be used for high-throughput sequencing;

Finally, the library was analyzed by bioinformatics to obtain gene mutation data.

Figure 2. Example of bioinformatics analysis process

Assemble the sequences with the same molecular tag (that is, the same sample source) into a unique template sequence

Detailed ways

In an embodiment of the present invention, a method for detecting gene mutation is provided, comprising the following steps:

A method for detecting gene mutations, comprising the following steps:

(1) Using the subject's plasma DNA as a template;

(2) Perform PCR pre-amplification;

(3) Carry out Index PCR secondary amplification to the pre-amplification product;

(4) Perform library quality control on the PCR secondary amplification products, and then perform 150bp paired-end sequencing on Illumina NextSeq;

(5) Remove the joints in the sequenced sequence, and then splice it into a sequence to obtain the original template sequence; compare the original template sequence with the human genome, and calculate the unique template sequence set by comparing the UMI molecular tag sequence of the original template sequence ;Use the unique template sequence to calculate the genome coverage for evaluating the specificity of the library; calculate the ratio of the mutant sequence to the reference sequence to calculate the somatic gene mutation rate in the detection region;

It is characterized by:

The primers used in the pre-amplification are the mixture of multiple pairs of primers, and the multiple pairs of primers are used to specifically amplify different sub-target regions in the region to be tested;

All forward primers contain linker sequence A, UMI molecular tag sequence and sub-target region-specific primer sequence from 5' end to 3' end;

All reverse primers contain linker sequence B, UMI molecular tag sequence and sub-target region-specific primer sequence from 5' end to 3' end;

The adapter sequences A in all primer pairs are identical to each other, the adapter sequences B are identical to each other, and the adapter sequences A and B are not identical;

The region to be tested comprises one or several specific gene sequence regions, and the sub-target region refers to DNA fragments in different regions in a specific gene.

Each pair of primers amplifies a sub-target region in the gene, and the UMI molecular tag sequences contained between the primer pairs are different, which is used to ensure that the UMI molecular tag sequences in the DNA amplification fragments derived from the same sub-target region are the same; and the source The UMI molecular tag sequences between the DNA amplification fragments in different sub-target regions are different from each other;

In the secondary amplification of the Index PCR, the secondary amplification primers used from the 3' end to the 5' end are the linker sequence and the universal sequencing linker sequence in turn, and one side of the primer is between the linker sequence and the universal sequencing linker sequence. There is also an Index sequence between them; the 3' ends of the forward primer and the reverse primer of the secondary amplification primer are respectively the adapter sequence A and the adapter sequence B; the Index sequence is used to mark different samples, Different samples use different Index sequences, and the secondary amplification primers for the same sample use the same Index sequence.

For the specific gene to be tested, those skilled in the art can design and amplify the sub-target region-specific primer sequence of the region according to the selected sub-target region, design or select a suitable linker sequence according to the general design rules of primers, and provide different The primer pair randomly designs the UMI molecular tag sequence, and then the pre-amplification primer set for the specific gene to be tested can be obtained. For example, it can be used to detect the genetic status of the fetus by cfDNA in the peripheral blood of pregnant women, such as single gene diseases such as deafness genes, and cancer detection by free DNA genes of tumor genes.

In some embodiments, the length of the linker sequence A and the linker sequence A is 8-15bp, and the length of the UMI molecular tag sequence is 5-8bp

In some embodiments, before performing the secondary amplification of the Index PCR, the DNA obtained by PCR pre-amplification is subjected to magnetic bead purification;

In some embodiments, after performing the Index PCR amplification, the amplified DNA is purified by magnetic beads.

The implementation of the present invention also includes providing a kit for detecting gene mutations, which is characterized in that it includes multiple primer pairs for specific pre-amplification of different sub-target regions in the region to be tested;

All forward primers contain linker sequence A, UMI molecular tag sequence and sub-target region-specific primer sequence from 5' end to 3' end;

All reverse primers contain linker sequence B, UMI molecular tag sequence and sub-target region-specific primer sequence from 5' end to 3' end;

The adapter sequences A in all primer pairs are identical to each other, the adapter sequences B are identical to each other, and the adapter sequences A and B are not identical;

The region to be tested comprises one or several specific gene sequence regions, and the sub-target region refers to different position regions in a specific gene;

Each pair of primers amplifies a sub-target region in the gene, and the UMI molecular tag sequences contained between the primer pairs are different, which is used to ensure that the UMI molecular tag sequences in the DNA amplification fragments derived from the same sub-target region are the same; and the source The UMI molecular tag sequences between the DNA amplified fragments in different sub-target regions are different from each other.

In some kits, secondary amplification primers for amplifying the pre-amplification products are also included, and the secondary amplification primers are adapter sequences, universal sequencing adapter sequences from the 3' end to the 5' end , wherein one side of the primer also includes an Index sequence between the adapter sequence and the universal sequencing adapter sequence; the 3' ends of the forward primer and the reverse primer of the secondary amplification primer are respectively the adapter sequence A and the Linker sequence B; the Index sequence is used to mark different samples, different samples use different Index sequences, and the secondary amplification primers for the same sample use the same Index sequence.

The 3' sequences of the forward and reverse primers are adapter sequence A and adapter sequence B respectively (the 5' end sequence of the secondary amplification primer is designed according to the library requirements of the Illumina sequencer)

In some kits, preferably, the length of the linker sequence A and the linker sequence A is 8-15 bp, and the length of the UMI molecular tag sequence is 5-7 bp.

The implementation of the present invention also includes providing a method for preparing a kit for detecting gene mutations, which is characterized in that it includes synthesizing and/or assembling a plurality of primer pairs with the following sequence characteristics,

All forward primers contain linker sequence A, UMI molecular tag sequence and sub-target region-specific primer sequence from 5' end to 3' end;

All reverse primers contain linker sequence B, UMI molecular tag sequence and sub-target region-specific primer sequence from 5' end to 3' end;

The adapter sequences A in all primer pairs are identical to each other, the adapter sequences B are identical to each other, and the adapter sequences A and B are not identical;

The region to be tested comprises one or several specific gene sequence regions, and the sub-target region refers to different position regions in a specific gene;

Each pair of primers amplifies a sub-target region in the gene, and the UMI molecular tag sequences contained between the primer pairs are different, which is used to ensure that the UMI molecular tag sequences in the DNA amplification fragments derived from the same sub-target region are the same; and the source The UMI molecular tag sequences between the DNA amplified fragments in different sub-target regions are different from each other.

In some preparation schemes, it also includes synthesizing and/or assembling secondary amplification primers for amplifying the pre-amplification products, and the adopted secondary amplification primers are sequentially from the 3' end to the 5' end Adapter sequence, universal sequencing adapter sequence, wherein one side primer also includes a section of Index sequence between the adapter sequence and the universal sequencing adapter sequence; the 3' ends of the forward primer and the reverse primer of the secondary amplification primer are respectively the The linker sequence A and the linker sequence B; the Index sequence is used to mark different samples, different samples use different Index sequences, and the secondary amplification primers for the same sample use the same Index sequence.

In some preparation schemes, the length of the linker sequence A and the linker sequence A is 8-15 bp, and the length of the UMI molecular tag sequence is 5-7 bp.

Application Example Detection of GJB2 and SLC26A4 Gene Mutations

GJB2 gene encodes gap junction protein 26, with a total length of 678bp and two exons. Its protein is expressed in the inner ear and plays an important role in maintaining the normal potential in the cochlea. Its gene mutation can lead to autosomal recessive hereditary deafness and autosomal dominant hereditary deafness, and the deafness caused by mutation accounts for about half of all autosomal recessive hereditary deafness, accounting for 60% of sporadic cases. The SLC26A4 gene is located on chromosome 7q22.3 with a full length of 2343bp. Its encoded protein pendrin is mainly expressed in the inner ear, thyroid and kidney, and its function is related to chloride ion and bicarbonate ion transport. Mutations in the SLC26A4 gene cause Pendred syndrome and DFNB4 nonsyndromic ears. Gao Z used SNPscan technology to screen 695 deaf patients in China and found that 38.7% of deaf patients were caused by mutations in the GJB2 and SLC26A4 genes, and found that the mutations in these genes showed an obvious founder effect, so the two genes caused The study of hereditary deafness has important clinical significance.

Therefore, there is an urgent need for a rapid and efficient method for detecting GJB2 and SLC26A4 gene mutations clinically. When the present inventor was studying the detection of fragmented DNA, he discovered a new method for detecting DNA fragments, that is, by adding molecular tags to the amplification primers, and then amplifying the DNA fragments. On this basis, combined with the second-generation high-throughput sequencing technology, the method was improved especially according to the GJB2 and SLC26A4 genes, and optimal primers were designed especially according to the GJB2 and SLC26A4 genes, and a pair of GJB2 and SLC26A4 was developed through plasma DNA. A method for sequencing and analyzing the mutation of SLC26A4 gene and a kit thereof.

There are only very few reports on the methodological research of non-invasive prenatal genetic diagnosis of hereditary deafness in the world. Due to the existence of multiple Indel variant sites in deafness hotspot variants, such as GJB2 (c.299delAT, c.235delC, c.176_191del16), there are still many problems in the cfDNA detection of different platform systems. For example, based on multiple platform systems such as SNP array, genome-wide SNP analysis of fetuses and parents is costly; conventional polymerase-based sequencing-by-synthesis is difficult to guarantee base accuracy, and will generate base-error sequencing noise, resulting in routine Library capture and high-throughput sequencing still cannot resolve biases in variation caused by amplification and sequencing; the newer cyclic single molecule amplification and resequencing technology (cSMART) technology is limited by its circular library efficiency and back-to-back primer site design Impact. The method of the present invention solves this problem very well. On the basis of conventional multiple amplification, UMI (Unique Molecular Identifier) molecular tags are added, and UMI is used to denoise the next-generation sequencing data, thereby performing low copy number variation detection.

Reliability of method validated by known templates for detection of GJB2 and SLC26A4 mutation sites

Cell line DNA: multiple DNA samples containing GJB2 and SLC26A4 gene hotspot mutations (all from the DNA sample bank of the First Affiliated Hospital of the Army Military Medical University) were ultrasonically disrupted, and 166±10bp fragments were recovered and mixed into normal human plasma DNA at a preset ratio Obtain the sample to be tested with a known mutation ratio (calculated according to the positive DNA concentration)):

The proportion of GJB2 gene hotspot mutations in this sample is expected to be as follows

mutation site expected mutation ratio c.235delC 5.50% c.299_300delAT 6.20% c.176_191del16 9.30%

The proportion of hot spot mutations in the SLC26A4 gene of this sample is expected to be as follows:

mutation site expected mutation ratio NM_000441:c.919-2A>G 2.70% NM_000441:c.2168A>G 4.50%

The sensitivity and stability of the method of the present invention were tested.

Pre-amplification primer MIX: Add the primers in the first group or the primers in the second group in Table 1, or all the primer sequences of the first and second groups into a 1.5ml EP tube

Table 1 Pre-amplification primer set

A mixture of 26 pairs of primers composed of primers shown in Seq ID No.1-46, 89-91, and 94-96, used to specifically amplify SNP sites and hotspot variations in different sub-target regions of the GJB2 gene;

A mixture of 23 pairs of primers composed of primers shown in Seq ID No.47-88, 92-93, and 97-98 is used to specifically amplify SNP sites and hotspot variations in different sub-target regions of the SLC26A4 gene;

"NNNNNN" in each primer is a synthetic A/G/C/T random sequence, which is UMI "uniquemolecular identifier". The UMIs in each pair of primers are different from each other.

Note: The 5'-end sequence "CCTACACGACGCTCTTCCGATCT" in the forward primer is the linker sequence A; the 5'-end sequence "TCAGACGTGTGCTCTTCCGATCT" in the reverse primer is the linker sequence B.3.PCR pre-amplification

Prepare the following reaction mixture

dna 24.8ul Buffer 10X 3ul dNTP (2.5mM) 1ul Primer Mix 1ul Taq polymerase 0.2ul 30ul

Reaction on the PCR instrument:

The recovered DNA samples were purified on Beckman magnetic beads and eluted in 25 μl of sterile dH20 or elution buffer. 4. Index PCR amplification

Table 2index primers

99 Primer For: 5-AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGC-3 100 Index Primer Rev: 5-CAAGCAGAAGACGGCATACGAGATCTCTTAATTGACTGGAGTTCAGACGTGTGCT-3

The 5' end of Primer F and the 5' end of Index Primer Rev are Illumina's public universal sequencing adapters; "CTCTTAAT" in the reverse primer is the Index sequence, which is used to distinguish different samples, and the 3' end sequence "CCTACACGACGC", Index Primer Rev 3' end "TCAGACGTGTGCT" is the 5' end sequence of adapter A and adapter B of the primers in Table 1.

Reaction on the PCR machine:

The recovered DNA samples were purified on Beckman magnetic beads and eluted in 25 μl of sterile dH20 or elution buffer to obtain the library.

After the prepared library passed the quality control of Qubit detection concentration, Illumina NextSeq performed 150bp paired-end sequencing (operation steps were performed according to the operation instructions of Illumina Company).

The sequence of high-throughput sequencing is spliced into a sequence according to the repeated region, the connection adapter is removed, the sequence is restored to the original template sequence, the template is compared to the human genome (hg19), and the unique sequence is calculated by comparing the molecular tag sequence of the template sequence. The set of template sequences, as shown in Figure 2: the sequences with the same molecular tags are assembled into a unique template sequence.

Utilizing unique template sequences, genome coverage is calculated for the assessment of library specificity and the statistical detection of regional somatic mutations by calculating the ratio of mutated sequences to reference sequences.

After amplification by traditional primers without molecular tags, sequencing cannot distinguish different sequences from the same source. By introducing UMI molecular tags and merging sequences with the same source UMI through bioinformatics analysis, the errors introduced by the DNA polymerase amplification process and the next-generation sequencing process are greatly reduced. Low copy number variants are detected accurately.

The detection results of hot spot mutation sites of GJB2 gene are as follows:

mutation site mutation ratio Test results c.235delC 5.59% check out c.299_300delAT 6.14% check out c.176_191del16 9.44% check out

The detection results of hot spot mutation sites of SLC26A4 gene are as follows:

mutation site mutation ratio Test results NM_000441:c.919-2A>G 2.87% check out NM_000441:c.2168A>G 4.56% check out

The above table shows the detection results of low-proportion hotspot mutations in the two genes, which are in line with the expected results.

At the same time, the detection results of the method of the present invention are also highly consistent with those of other methods. It shows that the method of the present invention can accurately detect low-copy mutations.

For the same sample, the ddPCR method was used to detect the mutation ratio of one of the sites, and the result was consistent with the detection result of the method of the present invention. However, ddPCR can only detect single-site mutations.

Under certain conditions, designing appropriate primers for different known mutations can directly achieve the purpose of distinguishing mutant and wild-type genes through PCR amplification combined with Sanger sequencing. This method is mainly for peripheral blood samples. PCR amplification is combined with detection of mutation types in peripheral blood samples for known sites, and then the method of the present invention is used for plasma mock DNA detection.

Finally, it is noted that the above embodiments are only used to illustrate the technical solutions of the present invention without limitation. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be carried out Modifications or equivalent replacements without departing from the spirit and scope of the technical solution of the present invention shall be covered by the claims of the present invention.

SEQUENCE LISTING

<110> The First Affiliated Hospital of the Army Medical University of the Chinese People's Liberation Army

<120> A method and kit for non-invasive detection of SLC26A4 gene mutation

<130> P1830402CN-CN-LJY-CQ-LSH

<160> 100

<170> PatentIn version 3.3

<210> 1

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223>rs7999318_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 1

cctacacgac gctcttccga tctnnnnnnna agtgacccgc tgtgcatgta t 51

<210> 2

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223>rs4769954_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 2

cctacacgac gctcttccga tctnnnnnna caggagatgc aggcagtcaa c 51

<210> 3

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223>rs9509170_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 3

cctacacgac gctcttccga tctnnnnnnng atcccatcag aaggaccctg t 51

<210> 4

<211> 54

<212>DNA

<213> Artificial sequence

<220>

<223>rs1537788_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 4

cctacacgac gctcttccga tctnnnnnnng agatgcttca aaatacagga atgg 54

<210> 5

<211> 54

<212>DNA

<213> Artificial sequence

<220>

<223>rs9552134_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 5

cctacacgac gctcttccga tctnnnnnnnt gcagagatga aattgtactt gcag 54

<210> 6

<211> 50

<212>DNA

<213> Artificial sequence

<220>

<223>rs12585595_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 6

cctacacgac gctcttccga tctnnnnnnnc cagccattct ctcccatgtt 50

<210> 7

<211> 55

<212>DNA

<213> Artificial sequence

<220>

<223>rs9509191_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 7

cctacacgac gctcttccga tctnnnnnnnt gagctacaat cgaactcatc tttcc 55

<210> 8

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223>rs11147616_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 8

cctacacgac gctcttccga tctnnnnnnnc tctgcttcag gggcttgttc t 51

<210> 9

<211> 52

<212>DNA

<213> Artificial sequence

<220>

<223>rs9552150_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 9

cctacacgac gctcttccga tctnnnnnnnc aggtaggacc caagatttgt gc 52

<210> 10

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223>rs9509098_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 10

cctacacgac gctcttccga tctnnnnnnng gagaagatgc ctcctccatt c 51

<210> 11

<211> 49

<212>DNA

<213> Artificial sequence

<220>

<223>rs870728_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 11

cctacacgac gctcttccga tctnnnnnnng actgcctgac ccctccaac 49

<210> 12

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223>rs35967187_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 12

cctacacgac gctcttccga tctnnnnnnng agtgaccacc ctggcacata a 51

<210> 13

<211> 52

<212>DNA

<213> Artificial sequence

<220>

<223>rs2313485_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 13

cctacacgac gctcttccga tctnnnnnnnc catcagatgt ggcattcaga aa 52

<210> 14

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223>rs7327952_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 14

cctacacgac gctcttccga tctnnnnnnna ccatcaccca tcacccctaa t 51

<210> 15

<211> 52

<212>DNA

<213> Artificial sequence

<220>

<223>rs8000719_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 15

cctacacgac gctcttccga tctnnnnnnng gttgtgacat aaacagggga ca 52

<210> 16

<211> 48

<212>DNA

<213> Artificial sequence

<220>

<223>rs9550644_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 16

cctacacgac gctcttccga tctnnnnnnnt gctgggcagg atggagac 48

<210> 17

<211> 50

<212>DNA

<213> Artificial sequence

<220>

<223>rs945368_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 17

cctacacgac gctcttccga tctnnnnnnng atttttctgg gtccccaagc 50

<210> 18

<211> 50

<212>DNA

<213> Artificial sequence

<220>

<223>rs9506430_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 18

cctacacgac gctcttccga tctnnnnnnnc agactgctca agccctctcc 50

<210> 19

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223>rs6490533_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 19

cctacacgac gctcttccga tctnnnnnnna ctctctgcgt gaggtgagtg g 51

<210> 20

<211> 50

<212>DNA

<213> Artificial sequence

<220>

<223>rs2872371_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 20

cctacacgac gctcttccga tctnnnnnnnc ggctctgtgc taggaaccag 50

<210> 21

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223>rs9552160_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 21

cctacacgac gctcttccga tctnnnnnnnt tggcacagat gcagacctct t 51

<210> 22

<211> 52

<212>DNA

<213> Artificial sequence

<220>

<223>rs9550642_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 22

cctacacgac gctcttccga tctnnnnnnng gaaccttgag tactgggctg aa 52

<210> 23

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223>rs7988514_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 23

cctacacgac gctcttccga tctnnnnnnnt ccctcaggtc ccatttagct c 51

<210> 24

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223>rs7999318_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 24

tcagacgtgt gctcttccga tctnnnnnnng agagtggaga gggagctgag g 51

<210> 25

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223>rs4769954_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 25

tcagacgtgt gctcttccga tctnnnnnnng ggacaggaga ccctgtgttt t 51

<210> 26

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223>rs9509170_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 26

tcagacgtgt gctcttccga tctnnnnnnng agtcccccttc tccctgaggt t 51

<210> 27

<211> 58

<212>DNA

<213> Artificial sequence

<220>

<223>rs1537788_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 27

tcagacgtgt gctcttccga tctnnnnnna aaacaaaata taagttttag gggctgaa 58

<210> 28

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223>rs9552134_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 28

tcagacgtgt gctcttccga tctnnnnnnng gtcttttgtg ggggttgaga c 51

<210> 29

<211> 53

<212>DNA

<213> Artificial sequence

<220>

<223>rs12585595_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 29

tcagacgtgt gctcttccga tctnnnnnnnc acagcagacc tcaacagaca gaa 53

<210> 30

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223>rs9509191_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 30

tcagacgtgt gctcttccga tctnnnnnnng ccctacactt ccattcccat c 51

<210> 31

<211> 47

<212>DNA

<213> Artificial sequence

<220>

<223>rs11147616_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 31

tcagacgtgt gctcttccga tctnnnnnnnc ctggccaaca accacca 47

<210> 32

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223>rs9552150_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 32

tcagacgtgt gctcttccga tctnnnnnnnt ttacaaatgc cagccaattc c 51

<210> 33

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223>rs9509098_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 33

tcagacgtgt gctcttccga tctnnnnnnnt ggttgtacgg gtattgcatg a 51

<210> 34

<211> 59

<212>DNA

<213> Artificial sequence

<220>

<223>rs870728_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 34

tcagacgtgt gctcttccga tctnnnnnnnt gtaatcacta tagacagacc cttttcagc 59

<210> 35

<211> 50

<212>DNA

<213> Artificial sequence

<220>

<223>rs35967187_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 35

tcagacgtgt gctcttccga tctnnnnnnng cgagggaggt ctcggataag 50

<210> 36

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223>rs2313485_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 36

tcagacgtgt gctcttccga tctnnnnnnnt ttccccaact attccacagc a 51

<210> 37

<211> 52

<212>DNA

<213> Artificial sequence

<220>

<223>rs7327952_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 37

tcagacgtgt gctcttccga tctnnnnnnng atcagcagtt gtcagaggct gt 52

<210> 38

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223>rs8000719_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 38

tcagacgtgt gctcttccga tctnnnnnnna aggaaaatgg tgcatgccta c 51

<210> 39

<211> 48

<212>DNA

<213> Artificial sequence

<220>

<223>rs9550644_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 39

tcagacgtgt gctcttccga tctnnnnnnng cgccacttcc ctgttgaa 48

<210> 40

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223>rs945368_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 40

tcagacgtgt gctcttccga tctnnnnnnng gcataggtgg attgccaatt t 51

<210> 41

<211> 52

<212>DNA

<213> Artificial sequence

<220>

<223>rs9506430_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 41

tcagacgtgt gctcttccga tctnnnnnnng aggttgctct aatcccacct ga 52

<210> 42

<211> 49

<212>DNA

<213> Artificial sequence

<220>

<223>rs6490533_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 42

tcagacgtgt gctcttccga tctnnnnnnnc tgcgtcaacg gctcctatg 49

<210> 43

<211> 49

<212>DNA

<213> Artificial sequence

<220>

<223>rs2872371_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 43

tcagacgtgt gctcttccga tctnnnnnnnc actggggaaa gggcaaatc 49

<210> 44

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223>rs9552160_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 44

tcagacgtgt gctcttccga tctnnnnnnnt ccaccaagag gaagaaaagg a 51

<210> 45

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223>rs9550642_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 45

tcagacgtgt gctcttccga tctnnnnnnnt cagaggacag ttccctgttg g 51

<210> 46

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223>rs7988514_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 46

tcagacgtgt gctcttccga tctnnnnnnnc cttttctttt gggcacgttt t 51

<210> 47

<211> 57

<212>DNA

<213> Artificial sequence

<220>

<223>rs28690459_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 47

cctacacgac gctcttccga tctnnnnnnna caaaagcctt cttaagttta ggaaaca 57

<210> 48

<211> 56

<212>DNA

<213> Artificial sequence

<220>

<223>rs73421853_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 48

cctacacgac gctcttccga tctnnnnnnnt ggtttaaagg agtatctgga gtcaaa 56

<210> 49

<211> 54

<212>DNA

<213> Artificial sequence

<220>

<223>rs17154507_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 49

cctacacgac gctcttccga tctnnnnnnt gacgttatca ggatacggct atct 54

<210> 50

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223>rs6944998_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 50

cctacacgac gctcttccga tctnnnnnnnc ctgggctcac ttgacacaaa c 51

<210> 51

<211> 59

<212>DNA

<213> Artificial sequence

<220>

<223>rs10276013_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 51

cctacacgac gctcttccga tctnnnnnnnc cacctattag cttctaaaac aatgtaagg 59

<210> 52

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223>rs7805114_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 52

cctacacgac gctcttccga tctnnnnnnna aaccagtcat gggccttagg a 51

<210> 53

<211> 56

<212>DNA

<213> Artificial sequence

<220>

<223>rs10242548_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 53

cctacacgac gctcttccga tctnnnnnnnt ggttacaaac ataaccctgt tattgc 56

<210> 54

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223>rs6949189_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 54

cctacacgac gctcttccga tctnnnnnnnt gcagacgggt ttgaaatttt g 51

<210> 55

<211> 59

<212>DNA

<213> Artificial sequence

<220>

<223>rs1008707_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 55

cctacacgac gctcttccga tctnnnnnnnt cactcaactc ttaaaagaat cctacttga 59

<210> 56

<211> 55

<212>DNA

<213> Artificial sequence

<220>

<223>rs78502829_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 56

cctacacgac gctcttccga tctnnnnnnnc atgacagcca ttctacagtt tatgc 55

<210> 57

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223>rs6466197_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 57

cctacacgac gctcttccga tctnnnnnnna ttgtccccag gccaataaac t 51

<210> 58

<211> 52

<212>DNA

<213> Artificial sequence

<220>

<223>rs10487275_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 58

cctacacgac gctcttccga tctnnnnnnna gggacaggat ccagtagaaa gg 52

<210> 59

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223>rs3823958_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 59

cctacacgac gctcttccga tctnnnnnnnc aaggtagcca gagaagggtg a 51

<210> 60

<211> 54

<212>DNA

<213> Artificial sequence

<220>

<223>rs6980029_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 60

cctacacgac gctcttccga tctnnnnnnnt gcacttaaag gtagaggctc agaa 54

<210> 61

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223>rs4730261_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 61

cctacacgac gctcttccga tctnnnnnnnc tctgaggatg gctatggctg t 51

<210> 62

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223>rs57592817_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 62

cctacacgac gctcttccga tctnnnnnnna ggctgccgta gatttggtct g 51

<210> 63

<211> 48

<212>DNA

<213> Artificial sequence

<220>

<223>rs2253856_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 63

cctacacgac gctcttccga tctnnnnnnng tgagccccagt cccctgaa 48

<210> 64

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223>rs982663_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 64

cctacacgac gctcttccga tctnnnnnnna gcattggctg agatccattg t 51

<210> 65

<211> 53

<212>DNA

<213> Artificial sequence

<220>

<223>rs6965933_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 65

cctacacgac gctcttccga tctnnnnnnng ctgttatctg ttccaaccaa gga 53

<210> 66

<211> 52

<212>DNA

<213> Artificial sequence

<220>

<223>rs2712208_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 66

cctacacgac gctcttccga tctnnnnnnng caggtcaaga caccaacaat ca 52

<210> 67

<211> 55

<212>DNA

<213> Artificial sequence

<220>

<223>rs12534598_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 67

cctacacgac gctcttccga tctnnnnnnnt caacaactta aatgtacacg cacag 55

<210> 68

<211> 53

<212>DNA

<213> Artificial sequence

<220>

<223>rs28690459_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 68

tcagacgtgt gctcttccga tctnnnnnnnt tgcctccagt tcactcctaa aag 53

<210> 69

<211> 53

<212>DNA

<213> Artificial sequence

<220>

<223>rs73421853_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 69

tcagacgtgt gctcttccga tctnnnnnnng ggggaaaata gacaagaaga agg 53

<210> 70

<211> 57

<212>DNA

<213> Artificial sequence

<220>

<223>rs17154507_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 70

tcagacgtgt gctcttccga tctnnnnnna acatttatca gtagttccaa gccaact 57

<210> 71

<211> 49

<212>DNA

<213> Artificial sequence

<220>

<223>rs6944998_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 71

tcagacgtgt gctcttccga tctnnnnnnnc ccacagatgc acccaagat 49

<210> 72

<211> 58

<212>DNA

<213> Artificial sequence

<220>

<223>rs10276013_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 72

tcagacgtgt gctcttccga tctnnnnnnng gatggagtgt aatagtatgg gattatgg 58

<210> 73

<211> 52

<212>DNA

<213> Artificial sequence

<220>

<223>rs7805114_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 73

tcagacgtgt gctcttccga tctnnnnnnng ctagtccatc aacttgcctt ca 52

<210> 74

<211> 52

<212>DNA

<213> Artificial sequence

<220>

<223>rs10242548_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 74

tcagacgtgt gctcttccga tctnnnnnnnc aatgggtcgc attagtcaaa ga 52

<210> 75

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223>rs6949189_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 75

tcagacgtgt gctcttccga tctnnnnnnnc ccttctccac cagactcaaa g 51

<210> 76

<211> 53

<212>DNA

<213> Artificial sequence

<220>

<223>rs1008707_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 76

tcagacgtgt gctcttccga tctnnnnnnnt tgagcttgag cacattacca aaa 53

<210> 77

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223>rs78502829_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 77

tcagacgtgt gctcttccga tctnnnnnnna gggtccaggc tggctactct a 51

<210> 78

<211> 55

<212>DNA

<213> Artificial sequence

<220>

<223>rs6466197_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 78

tcagacgtgt gctcttccga tctnnnnnnt gagtgttcag gttcaccaaa ttaca 55

<210> 79

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223>rs10487275_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 79

tcagacgtgt gctcttccga tctnnnnnnng gggagctttt tagggagagg a 51

<210> 80

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223>rs3823958_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 80

tcagacgtgt gctcttccga tctnnnnnnna aggggtggtc ttgctgacaa t 51

<210> 81

<211> 53

<212>DNA

<213> Artificial sequence

<220>

<223>rs6980029_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 81

tcagacgtgt gctcttccga tctnnnnnnng cctttgttca gcttgtatcc atc 53

<210> 82

<211> 50

<212>DNA

<213> Artificial sequence

<220>

<223>rs4730261_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 82

tcagacgtgt gctcttccga tctnnnnnnna ggcagtgctt gggtatgagg 50

<210> 83

<211> 52

<212>DNA

<213> Artificial sequence

<220>

<223>rs57592817_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 83

tcagacgtgt gctcttccga tctnnnnnnnt ccctaacact cagcacagtt gc 52

<210> 84

<211> 54

<212>DNA

<213> Artificial sequence

<220>

<223>rs2253856_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 84

tcagacgtgt gctcttccga tctnnnnnnnt catttttatc cactcctgca gtca 54

<210> 85

<211> 52

<212>DNA

<213> Artificial sequence

<220>

<223>rs982663_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 85

tcagacgtgt gctcttccga tctnnnnnnnt cgttgtgtgg gaaatattct gg 52

<210> 86

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223>rs6965933_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 86

tcagacgtgt gctcttccga tctnnnnnnna accacccccaa accctaagac a 51

<210> 87

<211> 52

<212>DNA

<213> Artificial sequence

<220>

<223>rs2712208_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 87

tcagacgtgt gctcttccga tctnnnnnnnc cacatcctgg aatcatttcc tc 52

<210> 88

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223>rs12534598_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 88

tcagacgtgt gctcttccga tctnnnnnnnt ttggggactg gtacgaaagg t 51

<210> 89

<211> 49

<212>DNA

<213> Artificial sequence

<220>

<223> chr13:20763486_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 89

cctacacgac gctcttccga tctnnnnnnnc tacttcccca tctcccaca 49

<210> 90

<211> 49

<212>DNA

<213> Artificial sequence

<220>

<223> chr13:20763421_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 90

cctacacgac gctcttccga tctnnnnnnnc tcccccttga tgaacttcc 49

<210> 91

<211> 49

<212>DNA

<213> Artificial sequence

<220>

<223> chr13:20763530_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 91

cctacacgac gctcttccga tctnnnnnnnt gggagatggg gaagtagtg 49

<210> 92

<211> 53

<212>DNA

<213> Artificial sequence

<220>

<223> chr7:107323898_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 92

cctacacgac gctcttccga tctnnnnnnnc aaggaattat taaaaccaat gga 53

<210> 93

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223> chr7:107350577_F

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 93

cctacacgac gctcttccga tctnnnnnnnt gtgatagaaa agctggagca a 51

<210> 94

<211> 49

<212>DNA

<213> Artificial sequence

<220>

<223> chr13:20763486_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 94

tcagacgtgt gctcttccga tctnnnnnnnc tacttcccca tctcccaca 49

<210> 95

<211> 48

<212>DNA

<213> Artificial sequence

<220>

<223> chr13:20763421_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 95

tcagacgtgt gctcttccga tctnnnnnnng ctcctagtgg ccatgcac 48

<210> 96

<211> 48

<212>DNA

<213> Artificial sequence

<220>

<223> chr13:20763530_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 96

tcagacgtgt gctcttccga tctnnnnnnna gatgagcagg ccgacttt 48

<210> 97

<211> 49

<212>DNA

<213> Artificial sequence

<220>

<223> chr7:107323898_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 97

tcagacgtgt gctcttccga tctnnnnnnnt tttccaggtt ggctccata 49

<210> 98

<211> 55

<212>DNA

<213> Artificial sequence

<220>

<223> chr7:107350577_R

<220>

<221> misc_feature

<222> (24)..(29)

<223> n is a, c, g, or t

<400> 98

tcagacgtgt gctcttccga tctnnnnnnng gttgaatatt taccgtttct aaaat 55

<210> 99

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223> Primer For

<400> 99

aatgatacgg cgaccaccga gatctacaca cactctttcc ctacacgacg c 51

<210> 100

<211> 55

<212>DNA

<213> Artificial sequence

<220>

<223> Index Primer Rev.

<400> 100

caagcagaag acggcatacg agatctctta attgactgga gttcagacgt gtgct 55

Claims (7)

1. A kit for non-invasive detection of SLC26A4 gene mutation, characterized in that, A mixture of 23 pairs of primers comprising primers shown in Seq ID No.47-88, 92-93, and 97-98 is used to specifically amplify different sub-target regions of the GJB2 gene; thereby simultaneously detecting the variation of the SLC26A4 gene. 2. kit according to claim 2, is characterized in that, also comprises the secondary amplification primer that is used to amplify described preamplification product, the forward and reverse primer of described secondary amplification primer is as Shown in Seq ID No.99 and 100. 3. The preparation method of the kit according to claim 1 or 2, characterized in that it comprises synthesizing and/or assembling primers of the nucleotide sequences shown in Seq ID No. 47-88, 92-93, 97-98. 4. preparation method according to claim 3, is characterized in that, also comprises the secondary amplification primer that synthesis and/or assembly are used for amplifying described pre-amplification product, the secondary amplification primer of described secondary amplification primer Forward and reverse primers are shown in Seq ID No.99 and 100. 5. A method for non-invasive detection of SLC26A4 gene mutation, comprising the following steps: (1) Using the subject's plasma DNA as a template; (2) Perform PCR pre-amplification; (3) Carry out Index PCR secondary amplification to the pre-amplification product; (4) Perform library quality control on the PCR secondary amplification products, and then perform 150bp paired-end sequencing on Illumina NextSeq; (5) Remove the linker from the sequenced sequence, and then splice it into a sequence to obtain the original template sequence, compare the original template sequence with the human genome, and calculate the unique template sequence set by comparing the UMI molecular tag sequence of the original template sequence ;Use the unique template sequence to calculate the genome coverage for evaluating the specificity of the library; calculate the ratio of the mutant sequence to the reference sequence to calculate the somatic gene mutation rate in the detection region; It is characterized by: In the pre-amplification, a mixture of 23 pairs of primers composed of primers shown in Seq ID No.47-88, 92-93, and 97-98 is used to simultaneously specifically amplify different sub-target regions of the SLC26A4 gene; thereby detecting SLC26A4 mutations ; The region to be tested includes one or several specific gene sequence regions, and the sub-target region refers to DNA fragments of different regions in a specific gene; Each pair of primers amplifies a sub-target region in the gene, and the UMI molecular tag sequences contained between the primer pairs are different to ensure that the UMI molecular tag sequences in the DNA amplification fragments derived from the same sub-target region are the same; and the source The UMI molecular tag sequences between the DNA amplification fragments in different sub-target regions are different from each other; In the secondary amplification of the Index PCR, the forward and reverse primers shown in Seq ID No.99 and 100 were used. 6. The method according to claim 5, characterized in that: before performing said Index PCR amplification, the DNA obtained by PCR pre-amplification is subjected to magnetic bead purification. 7. The method according to claim 5 or 6, characterized in that: after performing the Index PCR amplification, the amplified DNA is purified by magnetic beads.