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Methods for indexing samples and sequencing multiple polynucleotide templates

  • US 9,512,478 B2
  • Filed: 06/17/2014
  • Issued: 12/06/2016
  • Est. Priority Date: 02/02/2007
  • Status: Active Grant
First Claim
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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.

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