Methods for increasing yield of sequencing libraries
The present disclosure is concerned with compositions, methods, and kits for preparing a sequencing library. In one embodiment, methods include producing a library of target nucleic acids having the same adapter at each end and then switching the identity of one adapter to result in target nucleic acids flanked by distinct adapters.
1 . A method for producing a sequencing library comprising:
providing a plurality of double stranded symmetric modified target nucleic acids comprising a first adapter sequence at each end, wherein the first adapter sequence comprises a DNA lesion, with the proviso that the first adapter does not comprise a universal molecular identifier;
extending the modified target nucleic acids with a lesion-intolerant polymerase to generate a plurality of single stranded asymmetric modified target nucleic acids comprising at the 5′ end of each strand the first adapter sequence and at the 3′ end of each strand the complement of a portion of the first adapter;
annealing a primer to the plurality of single stranded asymmetric modified target nucleic acids, the primer comprising from 5′ to 3′ a second adapter sequence and an annealing domain, the annealing domain comprising a nucleotide sequence that anneals to the complement of the portion of the first adapter of the plurality of single stranded asymmetric modified target nucleic acids;
extending the 3′ end of the annealed asymmetric modified target nucleic acids with a lesion-intolerant polymerase using the primer as template,
wherein the extending results in a plurality of asymmetric modified target nucleic acids comprising from 5′ to 3′ (i) the first adapter, (ii) the target nucleic acid, (iii) the complement of the portion of the first adapter, and (iv) the complement of the second adapter.
2 . The method of claim 1 , wherein the plurality of symmetric modified target nucleic acids are double stranded and each strand comprises, from 5′ to 3′, the first adapter sequence comprising the DNA lesion, the target nucleic acid, a gap comprising at least one nucleotide, and the complement of a portion of the first adapter sequence without the DNA lesion.
3 . The method of claim 1 , wherein the DNA lesion comprises at least one of an abasic site, a modified base, a mismatch, a single-stranded break, or cross-linked nucleotides.
4 . The method of claim 1 , wherein the first adapter comprises one or more universal sequences, one or more index sequences, or a combination thereof.
5 . The method of claim 1 , wherein the target nucleic acids are from nucleic acids originating from a single cell, and wherein the nucleic acids comprise RNA or DNA.
6 . The method of claim 1 , wherein the target nucleic acids are from nucleic acids originating from a plurality of cells, and wherein the nucleic acids comprise RNA or DNA.
7 . The method of claim 6 , wherein the RNA comprises mRNA.
8 . The method of claim 6 , wherein the DNA comprises whole cell genomic DNA.
9 . The method of claim 4 , wherein the method comprises combinatorial indexing.
10 . The method of claim 1 , further comprising amplifying the asymmetric modified target nucleic acids,
wherein the amplifying comprises a second primer and a lesion-tolerant polymerase,
wherein the second primer comprises a nucleotide sequence that anneals to the first adapter sequence or the complement thereof.
11 . The method of claim 1 , wherein subsets of the plurality of asymmetric modified target nucleic acids are present in a plurality of compartments, wherein the compartments comprise a well or a droplet.
12 . The method of claim 1 , wherein the providing comprises (i) contacting a plurality of DNA fragments with the first adapter under conditions to ligate the first adapter to both ends of the DNA fragments, or (ii) contacting DNA with a transposome complex, wherein the transposome complex comprises a transposase and the first adapter, wherein the contacting occurs under conditions suitable for ligation of the first adapter into the DNA to generate the double stranded symmetric modified target nucleic acids.
13 . The method of claim 1 , further comprising:
providing a surface comprising a plurality of amplification sites,
wherein the amplification sites comprise at least two populations of attached single stranded capture oligonucleotides having a free 3′ end, and
contacting the surface comprising amplification sites with the plurality of asymmetric modified target nucleic acids under conditions suitable to produce a plurality of amplification sites that each comprise a clonal population of amplicons from an individual asymmetric modified target nucleic acid.
14 . The method of claim 1 , wherein the 3′ end of the primer is blocked.
15 . The method of claim 14 , wherein the 3′ end of the primer comprises a dideoxynucleotide.
16 . The method of claim 5 , wherein the RNA comprises mRNA.
17 . The method of claim 5 , wherein the DNA comprises whole cell genomic DNA.
18 . The method of claim 1 , wherein the annealing domain of the primer comprises at least one altered nucleotide that increases the melting temperature compared to the corresponding native DNA nucleotide.
19 . The method of claim 18 , wherein the at least one altered nucleotide comprises a locked nucleic acid or a bridged nucleic acid.