CN-121992074-A - Construction method and sequencing method of single-cell DNA library

Abstract

The invention provides a construction method and a sequencing method of a single-cell DNA library. The construction method comprises the steps of a) conducting single cell lysis, then mixing the single cell lysis with a first primer, conducting first amplification to obtain a first amplification product, b) conducting second amplification to obtain a single cell DNA library, wherein the first primer comprises a universal amplification fragment and a random amplification fragment, the second primer comprises an index fragment and a complementary fragment, the single cell DNA library is a library suitable for sequencing of a BGI platform or an Illumina platform, the corresponding universal amplification fragment is a fragment suitable for the BGI platform or the Illumina platform, the fragment suitable for the BGI platform is 8-9nt, and the fragment suitable for the Illumina platform is 10nt. The method can solve the problem of complex construction method of single-cell DNA library in the prior art, and is suitable for the field of single-cell sequencing.

Inventors

• ZHANG MINGYE
• WANG YOUYOU
• MA JIE
• HU YUGANG
• WU QIANG

Assignees

• 纳昂达(南京)生物科技有限公司

Dates

Publication Date

20260508

Application Date

20260114

Claims (10)

1. 1. A method of constructing a single cell DNA library, the method comprising: a) After single cell lysis, mixing the single cell with a first primer, and carrying out first amplification to obtain a first amplification product; b) Mixing the first amplification product with a second primer, and performing second amplification to obtain the single-cell DNA library; The first primer comprises a universal amplified fragment and a random amplified fragment, wherein the 3 'end of the universal amplified fragment is connected with the 5' section of the random amplified fragment; the second primer comprises an index fragment and a complementary fragment, wherein the 3 'end of the index fragment is connected with the 5' section of the complementary fragment; The single-cell DNA library is a library suitable for sequencing by a BGI platform or a library suitable for sequencing by an Illumina platform, Correspondingly, the universal amplified fragment in the first primer is a fragment suitable for a BGI platform or a fragment suitable for an Illumina platform; The length of the fragment suitable for the BGI platform is 8-9nt, and the length of the fragment suitable for the Illumina platform is 10nt.
2. 2. The method of claim 1, wherein the fragment suitable for use in the BGI platform has a sequence ATCCGACTT or TCCGACTT.
3. 3. The method according to claim 1, wherein the fragment suitable for the Illumina platform has the sequence shown in SEQ ID No. 2.
4. 4. The method according to claim 1, wherein the sequence of the randomly amplified fragment comprises a sequence of 2 to 4 NKK bases or a sequence of 6 to 8B bases.
5. 5. The construction method according to claim 2, wherein the sequence of the first primer suitable for the BGI platform is any one or more of the sequences shown in SEQ ID NOs 1,3 or 7-16; The sequence of the first primer suitable for the Illumina platform is shown as SEQ ID NO. 4.
6. 6. The method according to claim 1, wherein the first amplification is followed by first purification to obtain the first amplified product.
7. 7. The construction method according to claim 1, wherein the single-cell DNA library is obtained by performing a second purification after the second amplification.
8. 8. The method of any one of claims 1-7, wherein the first amplified system further comprises PEG and/or Mg 2+ ; Preferably, the PEG comprises PEG6000; Preferably, the concentration of PEG6000 in the first amplified system is 3-20 wt%, more preferably 5-15 wt%; Preferably, the concentration of Mg 2+ in the first amplified system is 5-10 nM, more preferably 6-9 nM; Preferably, the Mg 2+ is derived from MgCl 2 ; preferably, the first amplification comprises a gradient temperature-rising amplification.
9. 9. A single cell sequencing method is characterized in that, the single cell sequencing method comprises the following steps: constructing and obtaining the single-cell DNA library by using the construction method of the single-cell DNA library according to any one of claims 1 to 8; the single cell DNA library was sequenced using a high throughput sequencing platform.
10. 10. The single cell sequencing method of claim 9, wherein the high throughput sequencing platform comprises a BGI platform or an Illumina platform.

Description

Construction method and sequencing method of single-cell DNA library Technical Field The invention relates to the field of single-cell sequencing, in particular to a construction method and a sequencing method of a single-cell DNA library. Background Single cells are the basic functional units of life activities, the total amount of genomic DNA of human single cells is only about 6 pg, and the DNA of a typical single cell cannot directly meet the downstream experimental requirements (typically requiring μg-level DNA) such as whole genome sequencing due to the limitations of DNA library construction techniques. However, if a large amount of cell nucleic acid is directly mixed for library construction, genomic heterogeneity among cells (such as gene mutation differences of different cancer cells in a tumor) can be covered, or a large amount of cells cannot be obtained in a specific application scenario (such as chromosome abnormality detection of embryo cells and single cell sequencing of microorganisms). Therefore, single cell samples need to be "signal amplified" using single cell DNA amplification techniques to meet downstream detection requirements. Single-cell DNA amplification technology is a molecular biological technology that specifically amplifies trace amounts of genomic DNA at the single-cell level, amplifies minute amounts of single-cell genomic DNA to the microgram level, and then processes and analyzes the amplified DNA according to different requirements. The currently prevailing single-cell DNA amplification techniques are mainly based on the whole genome amplification (Whole Genome Amplification, WGA) principle and can be classified into three types according to amplification strategies, PCR-based, isothermal amplification (Multiple Displacement Amplification, MDA) and Multiple annealing circular amplification (Multiple ANNEALING AND Looping Based Amplification cycles, MALBAC). Subsequently, the amplified DNA was subjected to NGS library construction using an ultrasonic or enzymatic fragmentation protocol. Thus, the whole procedure of single-cell DNA detection scheme is longer, including single-cell DNA amplification and amplification product library construction. The PCR-based method combines random primers with genomic DNA, and amplification is achieved by a denaturation-annealing-extension cycle of PCR, a representative method being DOP-PCR (degenerate oligonucleotide primer PCR). Isothermal amplification method29 DNA polymerase (high fidelity, strong strand displacement activity) is used as a core, and the whole genome amplification is realized by combining genome DNA with random primers and continuously displacing and extending to form long-chain DNA. The multiple annealing circular amplification method combines DNA through special primer (random sequence at 3 'end and universal sequence at 5' end), forms circular DNA through multiple annealing extension, the amplification process is a semi-linear process, and finally amplified signal through universal primer. Three single-cell DNA amplification techniques each have advantages and disadvantages. The DOP-PCR method has the advantages of short reaction time, simple and convenient operation, better amplification uniformity, lower gene coverage, stronger preference, high MDA fidelity, long amplification fragments, high gene coverage, worse amplification uniformity, easy generation of chimerism, long reaction time, better MALDBAC method amplification uniformity, higher gene coverage, complex operation and higher cost. The core of the single-cell DNA amplification technology is to realize high-efficiency, uniform and high-fidelity whole genome amplification under a micro-template, and different methods (DOP-PCR, MDA, MALBAC) have adaptive scenes. However, each amplification method has the disadvantages, and no single-cell DNA amplification technology has the advantages of simple operation, short reaction time, high gene coverage, good amplification uniformity and high fidelity. In addition, the conventional single-cell DNA amplification technology needs a scheme of amplifying and then establishing a library, and has the advantages of complex operation flow and long time consumption. Disclosure of Invention The invention mainly aims to provide a construction method and a sequencing method of a single-cell DNA library, which are used for solving the problem that the construction method of the single-cell DNA library in the prior art is complex. In order to achieve the above object, according to a first aspect of the present invention, there is provided a construction method of a single-cell DNA library, comprising a) mixing a single-cell with a first primer after cleavage, performing a first amplification to obtain a first amplification product, b) mixing the first amplification product with a second primer, performing a second amplification to obtain the single-cell DNA library, wherein the first primer comprises a universal amplification frag