Method of constructing sequencing library
First Claim
1. A customized 5184-well plate-based method of constructing a sequencing library in a length ranging from 550 bp to 1000 bp, comprising steps of:
- 1) providing a single-stranded DNA fragment from a biological sample and distributing the single-stranded DNA fragments extracted from 10 to 500 cells into said 5184-well plate such that a probability of homologous chromosome fragments deriving from both mother and father at the same site in their respective genomes presenting in each well is less than 1%;
2) subjecting the single-stranded DNA fragment to whole genomic amplification to obtain a whole genome amplification product;
3) fragmenting the whole genome amplification product using a transposase embedded with two adaptors to obtain a fragmented product with two adaptors respectively at two ends; and
4) amplifying the fragmented product with two adaptors respectively at two ends using a tag sequence and a pair of primers to obtain said sequencing library,wherein each well of said 5184-well plate has a volume of 350 nL and an effective volume of 200 nL.
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Abstract
Provided is a method of constructing a sequencing library. The method includes 1) providing a single-stranded DNA fragment from a biological sample; 2) subjecting the single-stranded DNA fragment to whole genomic amplification to obtain a whole genome amplification product; 3) fragmenting the whole genome amplification product using a transposase embedded with two adaptors to obtain a fragmented product with two adaptors respectively at two ends; and 4) amplifying the fragmented product with two adaptors respectively at two ends using a tag sequence and a pair of primers to obtain said sequencing library.
6 Citations
18 Claims
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1. A customized 5184-well plate-based method of constructing a sequencing library in a length ranging from 550 bp to 1000 bp, comprising steps of:
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1) providing a single-stranded DNA fragment from a biological sample and distributing the single-stranded DNA fragments extracted from 10 to 500 cells into said 5184-well plate such that a probability of homologous chromosome fragments deriving from both mother and father at the same site in their respective genomes presenting in each well is less than 1%; 2) subjecting the single-stranded DNA fragment to whole genomic amplification to obtain a whole genome amplification product; 3) fragmenting the whole genome amplification product using a transposase embedded with two adaptors to obtain a fragmented product with two adaptors respectively at two ends; and 4) amplifying the fragmented product with two adaptors respectively at two ends using a tag sequence and a pair of primers to obtain said sequencing library, wherein each well of said 5184-well plate has a volume of 350 nL and an effective volume of 200 nL. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18)
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17. A method of constructing a sequencing library in a length ranging from 550 bp to 1000 bp using a customized 5184-well plate, comprising:
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1) providing a single-stranded DNA fragment from a biological sample; 2) distributing the single-stranded DNA fragments extracted from 10 to 500 cells into said 5184-well plate such that a probability of homologous chromosome fragments deriving from both mother and father at the same site in their respective genomes presenting in each well is less than 1%; 3) subjecting the single-stranded DNA fragment in each well of said 5184-well plate to whole genomic amplification to obtain a whole genome amplification product, wherein a reaction system in each well is in a volume less than 100 nL; 4) fragmenting the whole genome amplification product in each well of said 5184-well plate using a transposase embedded with two adaptors to obtain a fragmented product with two adaptors respectively at two ends; and 5) amplifying the fragmented product with two adaptors respectively at two ends in each well of said 5184-well plate using a tag sequence and a pair of primers to obtain said sequencing library, wherein the tag sequence comprises a first tag sequence and a second tag sequence, wherein the first tag sequence is composed of a third adaptor, a first random fragment and a first single-stranded DNA sequence in order from the 5′
end to the 3′
end; and
the second tag sequence is composed of a fourth adaptor, a second random fragment and a second single-stranded DNA sequence in order from the 5′
end to the 3′
end,wherein the first tag sequence comprises 72 tag sequences containing different said first random fragments respectively, and the second tag sequence comprises 72 tag sequences containing different said second random fragments respectively, wherein each well of said 5184-well plate has a volume of 350 nL and an effective volume of 200 nL.
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Specification