Compositions and methods for identification of a duplicate sequencing read
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 duplicate sequencing reads, the method comprising:
obtaining amplicons each comprising an amplified fragment of a nucleic acid with an appended adaptor, wherein each adaptor comprises an identifier site comprising a plurality of nucleotides unique to the amplified fragment;
sequencing the amplicons to generate sequence reads that include identifier and target sequences; and
identifying sequence reads with identical identifier and target sequences as duplicates.
2. The method of claim 1 , further comprising removing a duplicate read from the sequence reads.
3. The method of claim 1 , wherein the identifier site is sequenced with the target sequence.
4. The method of claim 1 , wherein the identifier site is sequenced separately from the target sequence.
5. The method of claim 1 , wherein the amplicons include amplified fragments of cDNA.
6. The method of claim 1 , wherein the amplicons include amplified fragments of genomic DNA.
7. The method of claim 1 , wherein each adaptor includes a primer binding site.
8. The method of claim 7 , wherein the primer binding site is a universal target sequencing primer binding site.
9. The method of claim 1 , wherein each unique identifier is at least 6 nucleotides in length.
10. The method of claim 1 , wherein the adaptors have been appended to the fragments using a ligation reaction or a priming reaction.
11. The method of claim 1 , wherein each unique identifier is at a junction between the adapter and the nucleic fragment.
12. The method of claim 1 , further comprising determining the genomic start position for each sequencing read.
13. The method of claim 1 , wherein each adaptor further comprises an indexing site.
14. The method of claim 1 , wherein each identifier site is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length.
15. The method of claim 1 , further comprising obtaining a sample comprising the nucleic acid, fragmenting the nucleic acid to yield the fragments of nucleic acid, appending the adaptors to the fragments of nucleic acid, and performing an amplification reaction to obtain the amplicons.
16. The method of claim 1 , wherein the amplicons include amplified fragment of a nucleic acid from a first sample with an appended adaptor that includes a first index and amplified fragment of a nucleic acid from a second sample with an appended adaptor that includes a second index, and the method includes separating sequence reads by sample based on the first or second index.