Compositions and methods for identification of a duplicate sequencing read
View Patent ↗The present invention provides methods, compositions and kits for detecting duplicate sequencing reads. In some embodiments, the duplicate sequencing reads are removed.
1. A method for detecting a duplicate sequencing read from a population of sample sequencing reads comprising:
a) ligating an adaptor to a 5′ end of each nucleic acid fragment of a plurality of nucleic acid fragments from one or more samples, wherein the adaptor comprises:
(i) an indexing primer binding site;
(ii) an indexing site;
(iii) an identifier site consisting of between 1 and 8 nucleotides; and
(iv) a target sequence primer binding site;
b) amplifying the adapter-nucleic acid fragment ligated products;
c) generating a population of sequencing reads from amplified adapter-nucleic acid fragment ligated products; and
d) identifying a duplicate sequencing read as the sequencing read comprising the same identifier site and nucleic acid fragment as another sequencing read in the population of sequencing reads;
wherein the indexing site is unique amongst a subset of the plurality of nucleic acid fragments and is an index for multiple polynucleotides; and wherein the sequence of the identifier site is variable in sequence content in a plurality of adaptors.
2. The method of claim 1 , wherein the method further comprises removing from the population of sequence reads the sequencing read with a duplicate identifier site and target sequence.
3. The method of claim 1 , wherein the identifier site is sequenced with the indexing site.
4. The method of claim 1 , wherein the adaptor comprises from 5′ to 3′:
(i) the indexing primer binding site;
(ii) the indexing site;
(iii) the identifier site; and
(iv) the target sequence primer binding site.
5. The method of claim 1 , wherein the plurality of nucleic acid fragments is generated from more than one sample.
6. The method of claim 5 , wherein the nucleic acid fragments from each sample have the same indexing site, and each sample has a unique indexing site.
7. The method of claim 6 , wherein the sequencing reads are separated based on the indexing site.
8. The method of claim 7 , wherein the separation of sequencing reads is performed prior to step d).
9. The method of claim 1 , wherein the nucleic acid fragments are DNA fragments, RNA fragments, or DNA/RNA fragments.
10. The method of claim 9 , wherein the nucleic acid fragments are genomic DNA fragments or cDNA fragments.
11. The method of claim 1 , wherein the indexing site is between 2 and 8 nucleotides in length.
12. The method of claim 1 , wherein the indexing site is about 6 nucleotides in length.
13. The method of claim 1 , wherein the identifier site is 6 nucleotides in length.
14. The method of claim 1 , wherein the identifier site is 8 nucleotides in length.
15. The method of claim 1 , wherein the indexing primer binding site is a universal indexing primer binding site.
16. The method of claim 1 , wherein the target sequence primer binding site is a universal target sequence primer binding site.
17. The method of claim 6 , wherein the method further comprises separating fragments or sequencing reads based on indexing site.
18. A method for detecting a duplicate sequencing read from a population of sample sequencing reads comprising:
a) ligating an adaptor to a 5′ end of each nucleic acid fragment of a plurality of nucleic acid fragments from one or more samples, wherein the adaptor comprises:
(i) an indexing primer binding site;
(ii) an indexing site;
(iii) an identifier site consisting of between 1 and 8 nucleotides; and
(iv) a target sequence primer binding site;
b) amplifying the adapter-nucleic acid fragment ligated products;
c) generating a population of sequencing reads from amplified adapter-nucleic acid fragment ligated products; and
d) identifying a duplicate sequencing read as the sequencing read comprising the same identifier site and nucleic acid fragment as another sequencing read in the population of sequencing reads;
wherein the indexing site is unique amongst a subset of the plurality of nucleic acid fragments and is an index for multiple polynucleotides; and wherein the sequence of the identifier site is not variable in sequence content in a plurality of adaptors.