Methods for indexing samples and sequencing multiple polynucleotide templates

摘要

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.

主权项

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.