Quality control method of single cell whole genome amplification product

Abstract

The invention relates to a method and a system for quality control of single cell whole genome amplification products. The method comprises detection of different chromosome segments. The invention also relates to reagents used in the method.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology, and in particular to quality control primers and methods for single cell whole genome amplification products. Background Art

[0002] "Single-cell whole-genome amplification" (WGA) is a technology for amplifying the whole genome at the single-cell level. Its principle is to amplify the trace whole-genome DNA of isolated single cells to obtain a complete genome with high coverage. Currently, the most common and widely used single-cell whole-genome amplification technologies are based on multiple annealing and looping-based amplification cycles (MALBAC), multiple displacement amplification (MDA) based on isothermal reaction, and primary template-directed amplification (PTA) combined with and specially modified on this basis.

[0003] The combination of single-cell whole genome amplification (WGA) and next-generation sequencing (NGS) is often used for genetic analysis in preimplantation genetic testing (PGT). However, before the next-generation library is constructed and loaded onto the machine, the fragment length, concentration, and integrity of the WGA product need to be quality controlled.

[0004] Therefore, using this efficient, simple, and reliable method to evaluate the quality of single-cell whole genome amplification products can effectively improve the success rate and quality of downstream genetic testing, reduce clinical testing costs and the economic burden on patients. There are still many problems with the quality control methods in the existing technology, such as being only applicable to certain single-cell whole genome amplification technologies, low detection sensitivity, and low accuracy. Summary of the invention

[0005] In response to the shortcomings of the existing technology, the inventors have developed a quality control method with high sensitivity and accuracy that is applicable to a variety of single-cell whole genome amplification products.

[0006] The first aspect of the present invention provides primers for amplifying mammalian cell DNA, wherein the amplification product of the primers comprises one or more fragments selected from the following:

[0007] The fragment of chrY amplified by using SEQ ID NO: 1 and 2 as primers,

[0008] The fragment of chr6 amplified by using SEQ ID NO:3 and 4 as primers,

[0009] The fragment of chrX amplified by primers SEQ ID NO: 5 and 6,

[0010] The fragment of chr2 amplified by primers SEQ ID NO: 7 and 8,

[0011] The fragment of chr15 amplified by using SEQ ID NO:9 and 10 as primers,

[0012] The fragment of chr4 amplified by using SEQ ID NO:11 and 12 as primers,

[0013] The fragment of chr18 amplified by using SEQ ID NO:13 and 14 as primers,

[0014] The fragment of chr3 amplified by primers SEQ ID NO: 15 and 16,

[0015] The fragment of chr1 amplified by using SEQ ID NO: 17 and 18 as primers,

[0016] A fragment of chr5 amplified using SEQ ID NO: 19 and 20 as primers.

[0017] In one or more embodiments, the primer is selected from:

[0018] (1) any one or more of the sequences shown in SEQ ID NOs: 1-20,

[0019] (2) a sequence that is at least 90% identical to (1); and

[0020] (3) A mixture of the sequences described in (1) and (2).

[0021] In one or more embodiments, the primer is selected from:

[0022] (1) a primer pair selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, and / or SEQ ID NO: 19 and SEQ ID NO: 20,

[0023] (2) a sequence that is at least 90% identical to (1); and

[0024] (3) A mixture of the sequences described in (1) and (2).

[0025] In one or more embodiments, the mammal is a human.

[0026] In one or more embodiments, the DNA is genomic DNA.

[0027] In one or more embodiments, the DNA is a single cell whole genome amplification product. The single cell whole genome amplification product is a MALBAC single cell whole genome amplification product, an MDA single cell whole genome amplification product, and / or a PTA single cell whole genome amplification product.

[0028] The present invention also provides a probe for detecting chromosome fragments of mammalian cells, comprising:

[0029] (1) A probe that recognizes one or more fragments selected from the following:

[0030] The fragment of chrY amplified by using SEQ ID NO: 1 and 2 as primers,

[0031] The fragment of chr6 amplified by using SEQ ID NO:3 and 4 as primers,

[0032] The fragment of chrX amplified by primers SEQ ID NO: 5 and 6,

[0033] The fragment of chr2 amplified by primers SEQ ID NO: 7 and 8,

[0034] The fragment of chr15 amplified by using SEQ ID NO:9 and 10 as primers,

[0035] The fragment of chr4 amplified by using SEQ ID NO:11 and 12 as primers,

[0036] The fragment of chr18 amplified by using SEQ ID NO:13 and 14 as primers,

[0037] The fragment of chr3 amplified by primers SEQ ID NO: 15 and 16,

[0038] The fragment of chr1 amplified by using SEQ ID NO: 17 and 18 as primers,

[0039] A fragment of chr5 amplified using SEQ ID NO: 19 and 20 as primers, and / or

[0040] (2) The complementary sequence of (1).

[0041] In one or more embodiments, the mammal is a human.

[0042] In one or more embodiments, the probe is complementary to the fragment described in (1).

[0043] The present invention also provides a kit for quality control of a single cell whole genome amplification product, which comprises (1) a primer for detecting chromosomes, and

[0044] Optional (2) probes for detecting chromosomes,

[0045] Wherein, the chromosomes include: chrY, chr6, chrX, chr2, chr15, chr4, chr18, chr3, chr1, chr5.

[0046] In one or more embodiments, the kit is used to perform quality control on single-cell whole genome amplification products.

[0047] In one or more embodiments, the kit comprises the primers described in any embodiment herein, and / or the probe described in any embodiment herein, and optionally further comprises reagents required for PCR.

[0048] In one or more embodiments, the mammal is a human.

[0049] In one or more embodiments, the single-cell whole genome amplification product is a MALBAC single-cell whole genome amplification product, an MDA single-cell whole genome amplification product, and / or a PTA single-cell whole genome amplification product.

[0050] The second aspect of the present invention relates to a method for quality control of single-cell whole genome amplification products, including detection of chromosome fragments.

[0051] In one or more embodiments, the detection of the chromosome fragment is performed by PCR amplification.

[0052] In one or more embodiments, the chromosomes include: chrY, chr6, chrX, chr2, chrl5, chr4, chrl8, chr3, chrl, and chr5.

[0053] In one or more embodiments, the detection result of the chromosome fragment is the size and / or number of the amplified fragment.

[0054] In one or more embodiments, the fragment comprises a nucleic acid sequence selected from one or more of the following:

[0055] The approximately 102 bp nucleic acid sequence of chrY amplified using SEQ ID NO: 1 and 2 as primers,

[0056] The approximately 155 bp nucleic acid sequence of chr6 amplified using SEQ ID NO: 3 and 4 as primers,

[0057] The approximately 213 bp nucleic acid sequence of chrX amplified using SEQ ID NO: 5 and 6 as primers,

[0058] The approximately 255 bp nucleic acid sequence of chr2 amplified using SEQ ID NO: 7 and 8 as primers,

[0059] The approximately 293 bp nucleic acid sequence of chr15 amplified using SEQ ID NO: 9 and 10 as primers,

[0060] The approximately 339 bp nucleic acid sequence of chr4 amplified using SEQ ID NO: 11 and 12 as primers,

[0061] The approximately 400 bp nucleic acid sequence of chr18 amplified using SEQ ID NO: 13 and 14 as primers,

[0062] The approximately 442 bp nucleic acid sequence of chr3 amplified using SEQ ID NO: 15 and 16 as primers,

[0063] The approximately 519 bp nucleic acid sequence of chr1 amplified using SEQ ID NO: 17 and 18 as primers,

[0064] The approximately 603 bp nucleic acid sequence of chr5 amplified using SEQ ID NO: 19 and 20 as primers.

[0065] In one or more embodiments, the PCR is performed using the primers described in any embodiment of the first aspect of this invention.

[0066] In one or more embodiments, the PCR is performed using a primer pair selected from SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, and / or SEQ ID NO: 19 and SEQ ID NO: 20.

[0067] The present invention also provides the use of the primers described in any embodiment of the present invention and optionally the probe described in any embodiment of the present invention in preparing a single cell whole genome amplification product quality control kit.

[0068] In one or more embodiments, the method for quality control of the single-cell whole genome amplification product includes: detecting amplification products of different chromosome fragments. In one or more embodiments, the single-cell whole genome amplification product quality control is a method for quality control of the single-cell whole genome amplification product as described in any embodiment of the present invention.

[0069] In one or more embodiments, the primers are used to detect the chromosome fragment by PCR amplification.

[0070] In one or more embodiments, the chromosomes comprise: chrY, chr6, chrX, chr2, chrl5, chr4, chrl8, chr3, chrl, and chr5.

[0071] In one or more embodiments, the detection result of the chromosome fragment is the size and / or number of the amplified fragment.

[0072] In one or more embodiments, the fragment comprises a nucleic acid sequence selected from one or more of the following:

[0073] The approximately 102 bp nucleic acid sequence of chrY amplified using SEQ ID NO: 1 and 2 as primers,

[0074] The approximately 155 bp nucleic acid sequence of chr6 amplified using SEQ ID NO: 3 and 4 as primers,

[0075] The approximately 213 bp nucleic acid sequence of chrX amplified using SEQ ID NO: 5 and 6 as primers,

[0076] The approximately 255 bp nucleic acid sequence of chr2 amplified using SEQ ID NO: 7 and 8 as primers,

[0077] The approximately 293 bp nucleic acid sequence of chr15 amplified using SEQ ID NO: 9 and 10 as primers,

[0078] The approximately 339 bp nucleic acid sequence of chr4 amplified using SEQ ID NO: 11 and 12 as primers,

[0079] The approximately 400 bp nucleic acid sequence of chr18 amplified using SEQ ID NO: 13 and 14 as primers,

[0080] The approximately 442 bp nucleic acid sequence of chr3 amplified using SEQ ID NO: 15 and 16 as primers,

[0081] The approximately 519 bp nucleic acid sequence of chr1 amplified using SEQ ID NO: 17 and 18 as primers,

[0082] The approximately 603 bp nucleic acid sequence of chr5 amplified using SEQ ID NO: 19 and 20 as primers.

[0083] A third aspect of the present invention relates to a method for amplifying chromosome fragments of a sample, wherein the chromosomes include: chrY, chr6, chrX, chr2, chr15, chr4, chr18, chr3, chr1, and chr5.

[0084] In one or more embodiments, the amplification is performed by PCR amplification.

[0085] In one or more embodiments, the PCR is performed using the primers described in any embodiment of the first aspect of this invention.

[0086] In one or more embodiments, the PCR is performed using a primer pair selected from SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, and / or SEQ ID NO: 19 and SEQ ID NO: 20. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1 It is the electrophoresis result of PCR reaction using the products of MALBAC amplification of 11 embryonic cell samples in Example 2 of the present invention as templates (the 11 embryonic cell samples are numbered 2, 3, 4, 5, 6, 7, 11, 12, 13, 14, and 15 respectively; M in the electrophoresis diagram is a DNA marker).

[0088] Figure 2 It is a graph showing the agarose electrophoresis results after amplification with 10 pairs of primers in Example 2 of the present invention (the 11 embryonic cell samples are numbered 2, 3, 4, 5, 6, 7, 11, 12, 13, 14, and 15, respectively; M in the electrophoresis graph is a DNA marker).

[0089] Figure 3 The products of the 11 embryonic cell samples amplified by MALBAC in Example 2 of the present invention are used as templates for second-generation sequencing. The sequencing results are the results of the chromosome copy number of the embryonic cell samples obtained by bioinformatics analysis. The ordinate in the figure represents the copy number (copy number), and the abscissa represents 22 chromosomes and 2 sex chromosomes (the corresponding results of the 11 embryonic cell samples are numbered RD-PGT-A2, RD-PGT-A3, RD-PGT-A4, RD-PGT-A5, RD-PGT-A6, RD-PGT-A7, RD-PGT-A11, RD-PGT-A12, RD-PGT-A13, RD-PGT-A14, and RD-PGT-A15, respectively).

[0090] Figure 4 It is the electrophoresis result of PCR reaction using the products of 9 embryonic cell samples amplified by MDA as template in Example 3 of the present invention (the 9 embryonic cell samples are numbered 81772, 81773, 81774, 81775, 81776, 81777, 81778, 81779, 81780 respectively; M in the electrophoresis diagram is DNA marker).

[0091] Figure 5 This is a graph of agarose electrophoresis results after amplification with 10 pairs of primers in Example 3 of the present invention (the 9 embryonic cell samples are numbered 81772, 81773, 81774, 81775, 81776, 81777, 81778, 81779, and 81780 respectively; M in the electrophoresis graph is a DNA marker).

[0092] Figure 6The products of the 9 embryonic cell samples amplified by MDA in Example 3 of the present invention are used as templates for next-generation sequencing. The sequencing results are the results of the chromosome copy number of the embryonic cell samples obtained by bioinformatics analysis. The ordinate in the figure represents the copy number, and the abscissa represents 22 chromosomes and 2 sex chromosomes (the corresponding result numbers of the 9 embryonic cell samples are RD-PGS-81772, RD-PGS-81773, RD-PGS-81774, RD-PGS-81775, RD-PGS-81776, RD-PGS-81777, RD-PGS-81778, RD-PGS-81779, and RD-PGS-81780, respectively).

[0093] Figure 7 The electrophoresis results of the products of the four embryonic cell samples after PTA amplification and the amplification by 10 pairs of quality control primers in Example 4 of the present invention (the left figure is the electrophoresis diagram of the PTA amplification product, the four embryonic cell samples are numbered 1, 2, 3, and 4, respectively, and M is a DNA marker in the electrophoresis diagram; the right figure is the electrophoresis diagram of PCR of the PTA amplification product by 10 pairs of quality control primers, the quality inspection results of the four embryonic cell samples are numbered 1, 2, 3, and 4, respectively, and M is a DNA marker in the electrophoresis diagram.

[0094] Figure 8 The products of the four embryonic cell samples amplified by PTA in Example 4 of the present invention are used as templates for next-generation sequencing. The sequencing results are the results of the chromosome copy number of the embryonic cell samples obtained through bioinformatics analysis. In the figure, the ordinate represents the copy number, and the abscissa represents 22 chromosomes and 2 sex chromosomes (the corresponding results of the four embryonic cell samples are numbered RD-PGT-A2, RD-PGT-A3, RD.SC1.PTA, RD.SC2.PTA, RD.SC3.PTA, and RD.SC4.PTA, respectively). DETAILED DESCRIPTION

[0095] The inventors have found that performing conventional agarose electrophoresis on the single-cell whole genome amplification products, analyzing their amplification efficiency and product length distribution range, and then analyzing the amplification of a group of genes distributed to different chromosome colors can improve the success rate of downstream CNV detection and the reliability of detection results, and reduce the invalidity of downstream genetic detection results and the economic losses caused by unqualified or abnormal WGA product quality.

[0096] Therefore, the inventors have conducted in-depth research and found that by detecting 10 chromosome fragments of chrY, chr6, chrX, chr2, chr15, chr4, chr18, chr3, chr1, and chr5, the quality of the amplified product of the whole genome of a single cell can be accurately controlled. The present invention is aimed at different single-cell whole genome amplification technologies: multiple displacement constant temperature amplification (MDA), multiple annealing loop amplification technology (MALBAC), and multiple displacement constant temperature amplification combined with specially modified primary template directional amplification technology (PTA), and developed 10 pairs of universal quality control primers, which are evenly distributed on autosomes and sex chromosomes, respectively located at chr1, chr2, chr3, chr4, chr5, chr6, chr15, chr18, chrX and chrY, and the quality control primers are more comprehensive, and the sensitivity of quality control is higher. The primers located at chrX and chrY have certain advantages in preventing the mixing of samples.

[0097] "Single-cell whole genome amplification" (WGA) is a technique for amplifying the whole genome at the single-cell level. Single-cell whole genome amplification techniques include, but are not limited to, degenerate oligonucleotide primer PCR (DOP-PCR), multiple displacement amplification (MDA), multiple annealing loop amplification (MALBAC), primary template directed amplification (PTA), etc. Those skilled in the art are aware of the principles and processes of these techniques.

[0098] Herein, "chr" refers to chromosome, and the number following it represents the chromosome number, for example, chr6 refers to chromosome 6. In one or more embodiments, the chromosome described herein refers to a human chromosome. Herein, the quality control of the single-cell whole genome amplification product is achieved by detecting a region of the chromosome or a chromosome fragment containing the region, the region including: nucleotides 2655130 to 2655231 of chrY; nucleotides 102503245 to 102503399 of chr6; nucleotides 40800346 to 40800558 of chrX; nucleotides 86259274 to 86259528 of chr2; nucleotides 86259274 to 86259528 of chr1 5 nucleotides 74703783 to 74704075; chr4 nucleotides 95185902 to 95186240; chr18 nucleotides 28611028 to 28611427; chr3 nucleotides 100466907 to 100467348; chr1 nucleotides 167849295 to 167849813; chr5 nucleotides 55206244 to 55206846. Herein, the reference chromosome version is GRCh37 / hg19.

[0099] The term "nucleic acid" or "polynucleotide" refers to a deoxyribonucleotide (DNA) or a ribonucleotide polymer (RNA) in single-stranded or double-stranded form, and its complement. Nucleic acids contain synthetic, non-natural or altered nucleotide bases. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms thereof. Examples of polynucleotides contemplated herein include single-stranded and double-stranded DNA, single-stranded and double-stranded RNA, and hybrid molecules having a mixture of single-stranded and double-stranded DNA and RNA. DNA can be a coding strand or a non-coding strand. In one or more embodiments, the sample comprises fragmented genomic DNA. Methods for obtaining genomic DNA and fragmenting are well known in the art.

[0100] The basic unit of DNA is deoxyribonucleotide, which is condensed into a long chain molecule through phosphodiester bonds. Each deoxyribonucleotide is composed of phosphate, deoxyribose and base. The bases (bp) of DNA mainly include adenine (A), guanine (G), cytosine (C) and thymine (T). In the double helix structure of double-stranded DNA, A and T are paired through hydrogen bonds, and G and C are paired through hydrogen bonds. DNA forms include cDNA, genomic DNA, fragmented DNA or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be of any length, such as 10-10kbp, 20-5kbp, 30-1kbp, 50-500bp, 100-400bp, 150-300bp or 200-250bp.

[0101] "Primer" as described herein refers to a nucleic acid molecule with a specific nucleotide sequence that guides synthesis at the start of nucleotide polymerization. It can be used as the starting point of template-guided DNA synthesis under suitable conditions (such as buffer and temperature) in the presence of four different nucleoside triphosphates and reagents for polymerization (such as DNA polymerase). The primer composition comprises one or more primers. Primers are usually two artificially synthesized oligonucleotide sequences, one primer is complementary to a DNA template strand at one end of the target region, and the other primer is complementary to another DNA template strand at the other end of the target region, and its function is to serve as the starting point of nucleotide polymerization. In vitro artificially designed primers are widely used in polymerase chain reaction (PCR), qPCR, sequencing and probe synthesis, etc. Primers can be of any length, such as 5-200bp, 10-100bp, 20-800bp or 25-50bp. In any given case, the length of the primer depends on, for example, the intended use of the primer, and is generally in the range of 15 to 30 nucleotides. Short primer molecules usually require lower temperatures to form sufficiently stable hybrid complexes with the template. Primers do not have to reflect the exact sequence of the template, but must be complementary enough to hybridize with the template. Primer sites are regions on the template that hybridize with primers. Those skilled in the art can design primers based on the common knowledge in the art according to the marker to be amplified (see, for example, PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratories, NY, 1995). In addition, some software packages for designing the best probes and / or primers used in various analyses are public, such as Primer3 available from the Center for Genome Research, Cambridge, Mass., USA. Obviously, its potential use should also be taken into account when designing probes or primers.

[0102] The primers of the present invention are used to detect different chromosome fragments. Exemplarily, the primers: (1) can amplify a fragment of chrY amplified by SEQ ID NOs: 1 and 2 as primers, (2) can amplify a fragment of chr6 amplified by SEQ ID NOs: 3 and 4 as primers, (3) can amplify a fragment of chrX amplified by SEQ ID NOs: 5 and 6 as primers, (4) can amplify a fragment of chr2 amplified by SEQ ID NOs: 7 and 8 as primers, (5) can amplify a fragment of chr15 amplified by SEQ ID NOs: 9 and 10 as primers, (6) can amplify a fragment of chr4 amplified by SEQ ID NOs: 11 and 12 as primers, (7) can amplify a fragment of chr18 amplified by SEQ ID NOs: 13 and 14 as primers, (8) can amplify a fragment of chr3 amplified by SEQ ID NOs: 15 and 16 as primers, (9) can amplify a fragment of chr4 amplified by SEQ ID NOs: 11 and 12 as primers, (10) can amplify a fragment of chr5 amplified by SEQ ID NOs: 11 and 12 as primers, (11) can amplify a fragment of chr6 amplified by SEQ ID NOs: 11 and 12 as primers, (12) can amplify a fragment of chr7 amplified by SEQ ID NOs: 11 and 12 as primers, (13) can amplify a fragment of chr8 amplified by SEQ ID NOs: 11 and 12 as primers, (14) can amplify a fragment of chr9 amplified by SEQ ID NOs: 11 and 12 as primer The fragment of chr1 amplified by primers SEQ ID NOs: 17 and 18, (10) can amplify the fragment of chr5 amplified by primers SEQ ID NOs: 19 and 20.

[0103] In one or more embodiments, the primer has: (1) a sequence shown in any one of SEQ ID NOs: 1-20, (2) a sequence having at least 90% identity with (1); or (3) a mixture of the sequences of (1) and (2). In one or more embodiments, the primer is a primer pair, and the primer pair has (1) a sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, and / or SEQ ID NO: 19 and SEQ ID NO: 20, (2) a sequence having at least 90% identity with (1); and (3) a mixture of the sequences of (1) and (2). When discussing primers, "recognition" as used herein means that the primers hybridize to the template sequence (e.g., under stringent or highly stringent conditions), and the fragments amplified by the primer pair include: (1) a fragment of chrY amplified by SEQ ID NOs: 1 and 2 as primers, (2) a fragment of chr6 amplified by SEQ ID NOs: 3 and 4 as primers, (3) a fragment of chrX amplified by SEQ ID NOs: 5 and 6 as primers, (4) a fragment of chr2 amplified by SEQ ID NOs: 7 and 8 as primers, (5) a fragment of chr15 amplified by SEQ ID NOs: 9 and 10 as primers, (6) a fragment of chr4 amplified by SEQ ID NOs: 11 and 12 as primers, (7) a fragment of chr18 amplified by SEQ ID NOs: 13 and 14 as primers, (8) a fragment of chr3 amplified by SEQ ID NOs: 15 and 16 as primers, (9) a fragment of chr4 amplified by SEQ ID NOs: 17 and 18 as primers, (10) a fragment of chr5 amplified by SEQ ID NOs: 19 and 20 as primers, (11) a fragment of chr6 amplified by SEQ ID NOs: 21 and 22 as primers, (12) a fragment of chr7 amplified by SEQ ID NOs: 23 and 24 as primers, (13) a fragment of chr8 amplified by SEQ ID NOs: 24 and 25 as primers, (14) a fragment of chr9 amplified by SEQ ID NOs: 25 and 26 as primers, (15) a fragment of chr10 amplified by SEQ ID NOs: 26 and The fragment of chr1 was amplified using primers NO:17 and 18, (10) the fragment of chr5 was amplified using primers SEQ ID NO:19 and 20.

[0104] As used herein, the term "hybridization" or "recognition" may refer to a process in which two single-stranded polynucleotides are non-covalently bound to form a stable double-stranded polynucleotide. In one aspect, the resulting double-stranded polynucleotide may be a "hybrid" or "double-stranded". The salt concentration in the "hybridization conditions" is generally less than about 1M, often less than about 500mM and may be less than about 200mM. "Hybridization buffer" includes a buffered salt solution, such as 5% SSPE, or other such buffers known in the art. The hybridization temperature may be as low as 5°C, but is generally higher than 22°C, and more generally higher than about 30°C, and generally exceeds 37°C. Hybridization is generally performed under stringent conditions, i.e., under which the sequence will hybridize with its target sequence but not with other non-complementary sequences. Stringent conditions depend on the sequence and vary in different situations. For example, longer fragments may require higher hybridization temperatures than short fragments for specific hybridization. Since other factors may affect the stringency of hybridization, including base composition and the length of the complementary chain, the presence of organic solvents, and the degree of base mismatching, the combination of parameters is more important than the absolute measurement of any one parameter alone. Typically stringent conditions are selected to be about 5°C lower than the melting temperature (Tm) of a specific sequence at a specific ionic strength and pH. Tm can be the temperature at which half of a population of double-stranded nucleic acid molecules are separated into single strands. Several equations for calculating the Tm of nucleic acids are well known in the art. As shown in standard references, when nucleic acids are in 1M NaCl aqueous solution, a simple estimated Tm value can be calculated by the formula Tm=81.5+0.41(%G+C) (see, e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985)). Other references (e.g., Allawi and SantaLucia, Jr., Biochemistry, 36:10581-94 (1997)) include alternative calculation methods that take into account structure and environment as well as sequence features when calculating Tm.

[0105] Typically, the stability of the hybrid is a function of ion concentration and temperature. Typically, the hybridization reaction is carried out under relatively low stringency conditions and then washed in a washing solution with different but higher stringency. Exemplary stringent conditions include pH about 7.0 to about 8.3, a temperature of at least 25°C, and a sodium ion (or other salt) concentration of at least 0.01M to no more than 1M. For example, 5xSSPE (750mM NaCl, 50mM sodium phosphate, 5mM EDTA, pH 7.4) and a temperature of about 30°C are suitable for allele-specific hybridization, although the appropriate temperature depends on the length and / or GC content of the hybridization region. In one aspect, the "hybridization stringency" for determining the percentage of mismatches can be as follows: 1) high stringency: 0.1x SSPE, 0.1% SDS, 65°C; 2) medium stringency (also referred to as moderate stringency): 0.2x SSPE, 0.1% SDS, 50°C; 3) low stringency: 1.0x SSPE, 0.1% SDS, 50°C. It should be understood that the same stringency can be achieved using alternative buffers, salts and temperatures. For example, moderate stringency hybridization can refer to conditions that allow nucleic acid molecules (e.g., probes) to bind to complementary nucleic acid molecules. The hybridized nucleic acid molecules typically have at least 60% identity, including, for example, at least 70%, 75%, 80%, 85%, 90% or 95% identity. Moderate stringency conditions can be conditions that achieve the same effect as the following conditions: 42°C, 50% formamide, 5x Denhardt solution, 5x SSPE, 0.2% SDS hybridization, followed by washing with 42°C, 0.2x SSPE, 0.2% SDS. High stringency conditions can be provided by, for example, 42°C, 50% formamide, 5x Denhardt solution, 5x SSPE, 0.2% SDS hybridization, followed by washing at 65°C, 0.1x SSPE and 0.1% SDS. Low stringency hybridization can be conditions that achieve equivalent results to the following conditions: 22°C, 10% formamide, 5x Denhardt's solution, 6x SSPE, 0.2% SDS hybridization, followed by washing in 1x SSPE, 0.2% SDS at 37°C. Denhardt's solution contains 1% polysucrose, 1% polyvinylpyrrolidone, and 1% bovine serum albumin (BSA). 20x SSPE (sodium chloride, sodium phosphate, EDTA) contains 3M sodium chloride, 0.2M sodium phosphate, and 0.025M EDTA.Other suitable moderate stringency and high stringency hybridization buffers and conditions are well known to those skilled in the art and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Plainview, NY (1989) and Ausubel et al., Short Protocols in Molecular Biology, 4th ed., John Wiley & Sons (1999).

[0106] The present invention can also use probes to detect the chromosome fragments described in the present invention. The "probe" described herein is a nucleic acid sequence (DNA or RNA) that recognizes a target sequence (complementary to the target sequence). The probe binds to the target gene through molecular hybridization to generate a hybridization signal, thereby displaying the target gene. The probe can include the entire target sequence or a fragment of the target sequence. The probe can be DNA or RNA transcribed therefrom. Usually, the probe carries a detection label, such as a fluorescent label. The fluorescent label includes but is not limited to FAM, CY5 and VIC. Fluorescent labels suitable for probes herein and methods of connecting them to probes are known in the art.

[0107] Exemplarily, the probe includes one or more selected from the following: (1) a probe that recognizes one or more fragments selected from the following: a fragment of chrY amplified using SEQ ID NOs: 1 and 2 as primers, a fragment of chr6 amplified using SEQ ID NOs: 3 and 4 as primers, a fragment of chrX amplified using SEQ ID NOs: 5 and 6 as primers, a fragment of chr2 amplified using SEQ ID NOs: 7 and 8 as primers, a fragment of chr15 amplified using SEQ ID NOs: 9 and 10 as primers, a fragment of chr4 amplified using SEQ ID NOs: 11 and 12 as primers, a fragment of chr18 amplified using SEQ ID NOs: 13 and 14 as primers, a fragment of chr3 amplified using SEQ ID NOs: 15 and 16 as primers, a fragment of chr1 amplified using SEQ ID NOs: 17 and 18 as primers, a fragment of chr5 amplified using SEQ ID NOs: 19 and 20 as primers, and / or (2) a complementary sequence of (1).

[0108] As used herein, the term "complementary" refers to hybridization or base pairing between nucleotides or nucleic acids, for example, between the two strands of a double-stranded DNA molecule, or between a primer binding site and an oligonucleotide primer on a single-stranded nucleic acid to be sequenced or amplified. Complementary nucleotides are typically A and T (or A and U), or C and G. Two single-stranded RNA or DNA molecules are said to be complementary when the nucleotides of one strand are optimally aligned, compared, and with appropriate nucleotide insertions or deletions, and are paired with at least about 80% (usually at least about 90% to 95%, more preferably about 98% to 100%) of the nucleotides of the other strand. Alternatively, complementarity exists when an RNA strand or DNA strand hybridizes to its complementary sequence under selective hybridization conditions. Typically, selective hybridization will occur when there is at least about 65% (preferably at least about 75%, more preferably at least about 90%) complementarity over a stretch of at least 14 to 25 nucleotides. See M. Kanehisa, Nucleic Acids Res. 12:203 (1984), incorporated herein by reference.

[0109] The present invention also provides a kit for detecting a single cell whole genome amplification product, which contains the primers described in any embodiment of the present invention, the probes described in any embodiment of the present invention, and the reagents required for PCR. Other reagents required for PCR, such as dNTP, buffer, MgSO4, polymerase, etc., are known in the art. Therefore, the present invention also includes the use of the primers described herein and the probes described herein in the preparation of a kit for quality control of a single cell whole genome amplification product.

[0110] On the other hand, the present invention provides a method for quality control of a single-cell whole genome amplification product, comprising detecting different chromosome fragments, wherein the chromosomes include: chrY, chr6, chrX, chr2, chr15, chr4, chr18, chr3, chr1, and chr5.

[0111] Methods for detecting chromosome fragments include, for example, using PCR amplification of cells or their DNA. Other reagents other than primers and / or probes required for chromosome fragment detection methods are known in the art. DNA sequencing includes first-generation, second-generation, and third-generation sequencing.

[0112] For example, chromosome fragments can be detected by the size and / or number of PCR amplified fragments. Exemplarily, the detection result of the chromosome fragment is the number of amplified fragments, wherein the fragment contains one or more nucleic acid sequences selected from the group consisting of: an approximately 102 bp nucleic acid sequence of chrY amplified by SEQ ID NOs: 1 and 2 as primers, an approximately 155 bp nucleic acid sequence of chr6 amplified by SEQ ID NOs: 3 and 4 as primers, an approximately 213 bp nucleic acid sequence of chrX amplified by SEQ ID NOs: 5 and 6 as primers, an approximately 255 bp nucleic acid sequence of chr2 amplified by SEQ ID NOs: 7 and 8 as primers, an approximately 293 bp nucleic acid sequence of chr15 amplified by SEQ ID NOs: 9 and 10 as primers, an approximately 339 bp nucleic acid sequence of chr4 amplified by SEQ ID NOs: 11 and 12 as primers, an approximately 400 bp nucleic acid sequence of chr18 amplified by SEQ ID NOs: 13 and 14 as primers, an approximately 442 bp nucleic acid sequence of chr3 amplified by SEQ ID NOs: 15 and 16 as primers, and an approximately 526 bp nucleic acid sequence of chr4 amplified by SEQ ID NOs: 17 and 18 as primers. The approximately 519 bp nucleic acid sequence of chr1 amplified using SEQ ID NOs: 17 and 18 as primers, and / or the approximately 603 bp nucleic acid sequence of chr5 amplified using SEQ ID NOs: 19 and 20 as primers.

[0113] Herein, the method for extracting DNA from a sample (eg, differentiated cells) is not particularly limited, and methods for extracting DNA suitable for use herein are well known in the art.

[0114] Advantages of the present invention:

[0115] 1. The method for quality control of MDA, MALBAC and PTA single-cell whole genome amplification products of the present invention is to perform conventional agarose electrophoresis on the single-cell whole genome amplification products, analyze the amplification efficiency and product length distribution range, and then analyze a group of gene amplifications distributed to different chromosome colors. The coverage of MDA, MALBAC and PTA amplification products is used to evaluate the uniformity of amplification, thereby improving the success rate of downstream CNV detection and the reliability of detection results, and reducing the invalidity of downstream genetic detection results and the economic losses caused by unqualified or abnormal WGA product quality.

[0116] 2. The 10 pairs of primers of the present invention contain gene fragments unique to Y staining, which can preliminarily distinguish the gender of the sample, thereby having certain advantages in preventing sample contamination in subsequent downstream experiments. Compared with other similar patents, there are many detection sites, and the genes detected by the 10 pairs of primers are evenly distributed, and can be carried out in the same reaction tube, which is simple and convenient to operate. At the same time, the requirements for the amount of product template are low, and the laboratory conditions are not high, and a conventional molecular laboratory can be used.

[0117] The present invention will be described below in the form of specific examples. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present invention. The materials, reagents and methods not specifically described in the examples are not conventional materials, reagents and methods in the art.

[0118] Example

[0119] Example 1: A quality control method for single cell whole genome amplification products

[0120] Ten pairs of primers (as shown in Table 1) were used to amplify gene fragments in conserved regions on ten different chromosomes. The experimental steps included the following steps:

[0121] 1) The 10 pairs of primer sequences used were purified by PAGE and freeze-dried into powder for storage and transportation;

[0122] 2) Dilute 10 pairs of primers into 100 μmol / L stock solutions with 1xTE buffer, then take 10 μl of the upstream and downstream primer stock solutions, mix them together and adjust the concentration to 10 μmol / L to prepare primer working solutions;

[0123] 3) Amplify the embryonic cell sample using a single-cell whole genome amplification kit, then take 200-300 ng of WGA amplification product and 1 μl of primer working solution, and use 2*KAPA multiple mix for PCR reaction;

[0124] 4) Take 1 μl of primer working solution and use 2*KAPA multiplex system for PCR reaction. The reaction system and thermal cycle conditions are as follows: 10 μl 2×KAPA mix, 9-x μl ddH2O, 200-300 ng template (volume x), mix well, and the total reaction volume is 20 μl. Denaturation at 96℃ for 3 min, 96℃ for 30 s → 62℃ for 30 s → 72℃ for 30 s, cycle 30 times, 72℃ for 2 min, 10℃∞;

[0125] 5) After the PCR reaction is completed, take 2μl of the product and add 2μl 2×DNA loading buffer to mix, then electrophoresed on a 2%-3% agarose gel at 120V for 30min, and then imaged with UV light on a gel imager; if the template DNA is of good quality, theoretically 9-10 products can be amplified; if there are only 8 or more amplified products, it means that the template DNA can be used for downstream library construction and CNV copy number detection; if there are less than 8 bands, it indicates that the quality of the template DNA is abnormal.

[0126] 6) The WGA amplification products after quality inspection are subjected to second-generation library construction and sequencing, and the chromosome copy number of the samples is analyzed by bioinformatics analysis on the second-generation data.

[0127] Table 1

[0128]

[0129] Embodiment 2: A method for Quality control method for single cell whole genome amplification products

[0130] Step 3) The single-cell whole genome amplification kit (Yikon) was used to amplify 11 embryonic cell samples, and the remaining steps were the same as in Example 1.

[0131] Figure 1 It is the result of electrophoresis of the products after MALBAC amplification of 11 embryonic cell samples. Figure 2 This is the agarose electrophoresis result after amplifying the WGA product with 10 pairs of primers. The quality inspection results show that the number of multiple quality inspection bands of the four embryo samples numbered 3, 7, 13 and 15 is less than 8, which are abnormal samples. Figure 3 The products of 11 embryonic cell samples amplified by MALBAC were used as templates for next-generation sequencing. The sequencing results were analyzed by bioinformatics to determine the chromosome copy number of the embryonic cell samples. Among them, the four embryonic samples with abnormal quality inspection, numbered 3, 7, 13 and 15, corresponded to Figure 3 The results showed that compared with the normal quality control samples, the chromosome copy number plots of the abnormal quality control embryo samples were abnormally scattered, and the results could not be judged. Therefore, the WGA product samples were subjected to multiple quality checks. If the number of bands was <8, the sample was abnormal and no downstream experimental tests were required. In addition, among the samples with normal WGA quality control, 5 samples (numbered 2, 5, 6, 11 and 12) had a 102bp product band located at chrY in all 10 pairs of primer quality controls ( Figure 2 ), indicating that these five samples are all male, which is consistent with the chromosome copy number results of these five embryonic cell samples, all of which have chrY copy number ( Figure 3 ); however, in the 10 primer pairs of the 2 samples (numbered 4 and 14), there was no 102 bp product band located at chrY ( Figure 2 ), indicating that both samples were female, which is consistent with the chromosome copy number results of bioinformatics analysis ( Figure 3 ), which also shows that these 10 pairs of primers have certain advantages in preventing contamination.

[0132] Example 3: A quality control method for MDA single cell whole genome amplification products

[0133] In step 3), 9 embryonic cell samples were amplified using REPLI-g Single Cell Kit (QIAGEN), and the remaining steps were the same as in Example 1.

[0134] Figure 4 It is the result of electrophoresis of the products after MDA amplification of 9 embryonic cell samples. Figure 5 This is the agarose electrophoresis result after amplifying the WGA product with 10 pairs of primers. The quality inspection results show that the number of multiple quality inspection bands of the three embryo samples numbered 81775, 81778 and 81780 are all <8, indicating that they are abnormal samples. Figure 6 The products of 9 embryonic cell samples amplified by MDA were used as templates for next-generation sequencing. The sequencing results were analyzed by bioinformatics to determine the chromosome copy number of the embryonic cell samples. Among them, the three embryonic samples with abnormal quality inspection numbers 81775, 81778 and 81780 corresponded to Figure 6 The results showed that compared with the normal samples, the chromosome copy number plots of the abnormal embryo samples were abnormally scattered, and the results could not be judged. Among the 6 samples with normal WGA quality control (numbered 81772, 81773, 81774, 81776, 81777, and 81779), only one sample 81777 had a 102bp product band located at chrY in the 10 primer pairs of quality control ( Figure 5 ), indicating that this sample is male, which is consistent with the chromosome copy number result of this embryonic cell sample, with chrY copy number ( Figure 6 ); while in 5 samples (numbered 81772, 81773, 81774, 81776, and 81779), there was no 102 bp product band located at chrY in the 10 pairs of primers ( Figure 6 ), indicating that these five samples are all female, which is consistent with the chromosome copy number results of bioinformatics analysis ( Figure 6 ).

[0135] Example 4: A quality control method for PTA single cell whole genome amplification products

[0136] In step 3), the four embryonic cell samples were amplified using the PTA single-cell whole genome amplification kit (Bioskryb), and the remaining steps were the same as in Example 1.

[0137] Figure 7The figure shows the electrophoresis results of the products of 4 embryonic cell samples after PTA amplification and the PTA products amplified with 10 pairs of primers. The quality inspection results show that the number of multiple quality inspection bands of the embryonic sample numbered 3 is less than 8, which is an abnormal sample. Figure 8 The products of 4 embryonic cell samples amplified by PTA were used as templates for next-generation sequencing. The sequencing results were analyzed by bioinformatics to determine the chromosome copy number of the embryonic cell samples. The abnormal embryo sample numbered 4 corresponds to Figure 8 The results showed that compared with the normal samples, the chromosome copy number plots of the abnormal embryo samples were abnormally scattered, and the results could not be accurately judged. In addition, the quality control results of the four samples showed that there was no 102bp product band located at chrY ( Figure 7 Right), indicating that these four samples are all female, which is consistent with the chromosome copy number results of these four embryonic cell samples, and there is no chrY copy number ( Figure 8 ).

Claims

1. A primer, wherein the amplification product of the primer comprises one or more fragments selected from the following: a fragment of chrY amplified by using SEQ ID NOs: 1 and 2 as primers, a fragment of chr6 amplified by using SEQ ID NOs: 3 and 4 as primers, a fragment of chrX amplified by using SEQ ID NOs: 5 and 6 as primers, a fragment of chr2 amplified by using SEQ ID NOs: 7 and 8 as primers, a fragment of chr15 amplified by using SEQ ID NOs: 9 and 10 as primers, a fragment of chr4 amplified by using SEQ ID NOs: 11 and 12 as primers, a fragment of chr18 amplified by using SEQ ID NOs: 13 and 14 as primers, a fragment of chr3 amplified by using SEQ ID NOs: 15 and 16 as primers, a fragment of chr1 amplified by using SEQ ID NOs: 17 and 18 as primers, and a fragment of chr5 amplified by using SEQ ID NOs: 19 and 20 as primers; Preferably, the primers are used to amplify single cell whole genome amplification products. More preferably, the primer comprises: (1) a sequence shown in any one or more of SEQ ID NOs: 1-20, and / or (2) a sequence having at least 90% identity with (1).

2. A probe capable of identifying: (1) One or more fragments selected from the following: The fragment of chrY amplified by using SEQ ID NO: 1 and 2 as primers, The fragment of chr6 amplified by using SEQ ID NO:3 and 4 as primers, The fragment of chrX amplified by primers SEQ ID NO: 5 and 6, The fragment of chr2 amplified by primers SEQ ID NO: 7 and 8, The fragment of chr15 amplified by using SEQ ID NO:9 and 10 as primers, The fragment of chr4 amplified by using SEQ ID NO:11 and 12 as primers, The fragment of chr18 amplified by using SEQ ID NO:13 and 14 as primers, The fragment of chr3 amplified by primers SEQ ID NO: 15 and 16, The fragment of chr1 amplified by using SEQ ID NO: 17 and 18 as primers, A fragment of chr5 amplified using SEQ ID NO: 19 and 20 as primers, and / or (2) The complementary sequence of (1).

3. The probe according to claim 2, characterized in that The probe is complementary to the fragment described in (1).

4. A kit comprising the primers according to claim 1 and / or the probe according to claim 2, and optionally further comprising reagents required for PCR.

5. A method for quality control of a single cell whole genome amplification product, comprising detecting fragments of different chromosomes, wherein the chromosome selection comprises: chrY, chr6, chrX, chr2, chr15, chr4, chr18, chr3, chr1, and chr5.

6. The method according to claim 5, characterized in that The detection of the chromosome fragment is performed by PCR amplification, and / or The detection result of the chromosome fragment is the size and / or number of the amplified fragment, and / or The single-cell whole genome amplification product is a MALBAC single-cell whole genome amplification product, an MDA single-cell whole genome amplification product, and / or a PTA single-cell whole genome amplification product.

7. The method according to claim 6, characterized in that The fragment comprises a nucleic acid sequence selected from one or more of the following: an approximately 102 bp nucleic acid sequence of chrY amplified using SEQ ID NOs: 1 and 2 as primers, an approximately 155 bp nucleic acid sequence of chr6 amplified using SEQ ID NOs: 3 and 4 as primers, an approximately 213 bp nucleic acid sequence of chrX amplified using SEQ ID NOs: 5 and 6 as primers, an approximately 255 bp nucleic acid sequence of chr2 amplified using SEQ ID NOs: 7 and 8 as primers, an approximately 293 bp nucleic acid sequence of chr15 amplified using SEQ ID NOs: 9 and 10 as primers, an approximately 339 bp nucleic acid sequence of chr4 amplified using SEQ ID NOs: 11 and 12 as primers, an approximately 400 bp nucleic acid sequence of chr18 amplified using SEQ ID NOs: 13 and 14 as primers, an approximately 442 bp nucleic acid sequence of chr3 amplified using SEQ ID NOs: 15 and 16 as primers, an approximately 526 bp nucleic acid sequence of chr4 amplified using SEQ ID NOs: 17 and 18 as primers, an approximately 512 bp nucleic acid sequence of chr5 amplified using SEQ ID NOs: 19 and 20 as primers, an approximately 256 bp nucleic acid sequence of chr6 amplified using SEQ ID NOs: 21 and 25 The approximately 519 bp nucleic acid sequence of chr1 was amplified using SEQ ID NOs: 17 and 18 as primers, and the approximately 603 bp nucleic acid sequence of chr5 was amplified using SEQ ID NOs: 19 and 20 as primers.

8. Use of the primers according to claim 1 and / or the probes according to claim 2 in preparing a kit for quality control of single cell whole genome amplification products, Preferably, the quality control includes genetic testing of different chromosomes. Preferably, the chromosome is selected from one or more of the following: chrY, chr6, chrX, chr2, chrl5, chr4, chrl8, chr3, chrl, chr5.

9. The use according to claim 8, characterized in that The single-cell whole genome amplification product is a MALBAC single-cell whole genome amplification product, an MDA single-cell whole genome amplification product, and / or a PTA single-cell whole genome amplification product.

10. A method for amplifying a chromosome fragment of a sample, wherein the chromosome is selected from one or more of the following: chrY, chr6, chrX, chr2, chr15, chr4, chr18, chr3, chr1, chr5, and the amplification is performed using the primers described in claim 1.

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