Methods for indexing samples and sequencing multiple polynucleotide templates
The invention relates to methods for indexing samples during the sequencing of polynucleotide templates, resulting in the attachment of tags specific to the source of each nucleic acid sample such that after a sequencing run, both the source and sequence of each polynucleotide can be determined. Thus, the present invention pertains to analysis of complex genomes (e.g., human genomes), as well as multiplexing less complex genomes, such as those of bacteria, viruses, mitochondria, and the like.
1. A method for sequencing nucleic acid sequences and identifying subsets of nucleic acid sequences, each subset of nucleic acid sequences isolated from a different source, the method comprising the steps of:
a) providing at least two different samples comprising nucleic acid target sequences and amplifying the target sequences with two or more sample specific amplification primers to generate amplified adaptor-target-adaptors, wherein one of said amplification primers comprises a sample specific tag sequence, and wherein said sample specific tag sequence differentiates amplified adaptor-target-adaptors originating from different samples;
b) pooling the amplified adaptor-target-adaptors of the at least two different samples;
c) immobilizing the pooled adaptor-target-adaptors on a surface;
d) sequencing the immobilized adaptor-target-adaptors on the surface to determine a sequence read of each immobilized adaptor-target-adaptor by hybridizing a first sequencing primer to the immobilized adaptor-target-adaptors, performing a first sequencing read and removing the first sequencing primer;
e) sequencing the sample specific tag sequence of each immobilized adaptor-target-adaptor by hybridizing a second sequencing primer to the immobilized adaptor-target-adaptors and performing a second sequencing read, wherein steps d) and e) determine nucleic acid sequences of the immobilized adaptor-target-adaptors and identify each of the immobilized adaptor-target-adaptors as a member of a subset of nucleic acid sequences.
2. The method of claim 1 , wherein amplifying in step (a) comprises nested PCR amplification.
3. The method of claim 1 , wherein step (d) occurs after step (e).
4. The method of claim 1 , wherein one or more of said amplification primers comprises a binding site for a sequencing primer.
5. The method of claim 1 , further comprising amplifying the immobilized adaptor-target-adaptors after step (c).
6. The method of claim 1 , wherein the immobilized adaptor-target-adaptors undergo a sequencing reaction from both ends to obtain a paired end read.
7. The method of claim 6 , wherein the sequence read of step (d) is obtained from one end of the immobilized adaptor-target-adaptors.
8. The method of claim 7 , further comprising synthesizing a complementary copy of the immobilized adaptor-target-adaptors and sequencing the opposite end of the complementary copy.
9. The method of claim 8 , wherein the opposite end of the complementary copy is sequenced before step (e).
10. The method of claim 8 , wherein the opposite end of the complementary copy is sequenced after step (e).
11. The method of claim 1 , wherein one or more amplification primers comprises the sequence of any one of SEQ ID NOs: 3-20.
12. The method of claim 1 , wherein one or more amplification primers comprises the sequence or complement of SEQ ID NO: 55 or 56.
13. The method of claim 1 , wherein one or more amplification primers comprises the sequence or complement of SEQ ID NO: 57.
14. The method of claim 1 , wherein one or more amplification primers comprises the sequence or complement of SEQ ID NO: 58.
15. The method of claim 1 , wherein one or more amplification primers comprises the sequence or complement of SEQ ID NO: 61.
16. The method of claim 1 , wherein one or more amplification primers comprises the sequence or complement of SEQ ID NO: 62.
17. The method of claim 1 , wherein one or more amplification primers comprises the sequence or complement of SEQ ID NO: 63.