Construction method for high-throughput sequencing library, kit, and use

Abstract

Embodiments provide a construction method for a high-throughput sequencing library, a kit, and a use. The method comprises: using a Tn5 transposase complex to randomly break a DNA in a genome sample to obtain a DNA breakage product; carrying out molecular hybridization on an RNA enrichment probe and the DNA breakage product, and using streptavidin magnetic beads to collect an RNA-DNA complex in a molecular hybridization product; and carrying out methylation treatment and PCR amplification on the RNA-DNA complex to obtain a sequencing library. The method achieves directed enrichment of a target methylation site in the genome sample; when a new target methylation site needs to be added, an RNA enrichment probe is simply designed for same; when the target methylation site that has been proven to have a poor effect needs to be deleted, the RNA enrichment probe corresponding thereto is simply removed; and by means of the directed enrichment of the target methylation site, the concentration requirement of the genome sample is reduced, and trace detection is facilitated.

Claims

1. A method for constructing a high-throughput sequencing library, characterized in that: include: The DNA in the genomic sample is randomly fragmented using the Tn5 transposase complex to obtain DNA fragmentation products, wherein the Tn5 transposase complex is embedded with an adapter sequence for sequencing; performing molecular hybridization on an RNA enrichment probe and the DNA fragmentation product, and collecting RNA-DNA complexes in the molecular hybridization product using streptavidin magnetic beads, wherein the RNA enrichment probe is a biotin-labeled RNA probe sequence, and the RNA probe sequence includes a target methylation site binding sequence; methylating the RNA-DNA complex to obtain a methylated product; The methylation products are amplified by PCR to obtain a sequencing library.

2. The method for constructing a high-throughput sequencing library according to claim 1, wherein The DNA template of the RNA probe sequence includes the nucleotide sequence shown in SEQ ID NO: 1, the target methylation site sequence and the nucleotide sequence shown in SEQ ID NO:

2.

3. The method for constructing a high-throughput sequencing library according to claim 1, wherein The target methylation site is selected from the group consisting of an EZH2 gene regulatory site, a SUZ12 gene regulatory site, and a REST gene regulatory site; The target methylation site is located in the non-coding region of FHL2, OTUD7A gene, CLORF132 gene, CCDC102B gene, ELOVL2 gene, EDARADD gene, TOM1L1 gene, NPTX2 gene or PRC2 complex binding protein gene sequence.

4. The method for constructing a high-throughput sequencing library according to claim 1, wherein The Tn5 transposase complex includes Tn5 transposase and a junction fragment bound to the Tn5 transposase, wherein the junction fragment includes the linker sequence.

5. The method for constructing a high-throughput sequencing library according to claim 4, wherein: The Tn5 transposase complex includes a first joining fragment and a second joining fragment, wherein the first joining fragment includes the nucleotide sequence shown in SEQ ID NO: 5 and the nucleotide sequence shown in SEQ ID NO: 3 in sequence; the second joining fragment includes the nucleotide sequence shown in SEQ ID NO: 5 and the nucleotide sequence shown in SEQ ID NO: 4 in sequence.

6. The method for constructing a high-throughput sequencing library according to claim 1, wherein: The methylation product is subjected to PCR amplification to obtain a sequencing library, comprising: The methylation product is amplified by PCR using library construction primers containing a barcode sequence to obtain the sequencing library, and the barcode sequence is used to identify the genomic sample.

7. The method for constructing a high-throughput sequencing library according to claim 6, wherein: The library construction primers include the nucleotide sequence shown in SEQ ID NO: 6 and the nucleotide sequence shown in SEQ ID NO: 7 or SEQ ID NO:

8.

8. A method for detecting epigenetic modification, characterized in that: include: Obtaining a sequencing library according to the method for constructing a high-throughput sequencing library according to any one of claims 1 to 7; The sequencing library is subjected to high-throughput sequencing to obtain methylation information of the genomic sample.

9. A biological age prediction method, characterized in that: include: Obtaining methylation information of a genomic sample according to the method for detecting epigenetic modification according to claim 8; Obtaining the methylation rate of each target methylation site according to the methylation information; The methylation rate of the target methylation site is input into a pre-trained biological age clock model, and the corresponding predicted age is output.

10. The biological age prediction method according to claim 9, characterized in that: The training steps of the biological age clock model include: Obtaining a training sample, wherein the training sample includes methylation rates and actual ages of multiple target methylation sites, wherein the read coverage of the target methylation sites is greater than a preset coverage threshold; Inputting the training sample into a penalized regression model to output a predicted age calculated based on the methylation rates of multiple target methylation sites in the training sample; According to the comparison result between the predicted age and the actual age, the parameters of the penalty regression model are adjusted until the penalty regression model converges to obtain the biological age clock model.

11. A high-throughput sequencing library construction kit, characterized in that: include: A Tn5 transposase complex for randomly fragmenting DNA in a genomic sample, wherein the Tn5 transposase complex is embedded with an adapter sequence for sequencing and a barcode sequence for identifying the genomic sample; An RNA enrichment probe for molecular hybridization with the DNA fragmentation product, wherein the RNA enrichment probe is a biotin-labeled RNA probe sequence, and the RNA probe sequence includes a target methylation site binding sequence; Streptavidin magnetic beads for collecting RNA-DNA complexes in molecular hybridization products; Methylation reagents for methylation of RNA-DNA complexes; and library construction primers for PCR amplification of the methylation products.

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