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Method for accurately counting starting molecules

  • US 8,715,967 B2
  • Filed: 03/29/2013
  • Issued: 05/06/2014
  • Est. Priority Date: 09/21/2010
  • Status: Active Grant
First Claim
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1. A method for estimating the number of initial target nucleic acid molecules in a sample, comprising:

  • a) amplifying a population of initial target nucleic acid molecules from a tagged sample thereby producing a population of amplified target DNA molecules, wherein the initial target nucleic acid molecules that comprise a target region are tagged with different degenerate base region (DBR) sequences, wherein said DBR sequences comprise at least one nucleotide base selected from;

    R, Y, S, W, K, M, B, D, H, V, N and modified versions thereof and wherein the amplified target DNA molecules comprise said target region and an associated DBR sequence of said DBR sequences;

    b) sequencing a plurality of the amplified target DNA molecules, thereby producing a plurality of sequence reads, wherein the sequencing step provides, for each of the amplified target DNA molecules that are sequenced, the nucleotide sequence of;

    (i) at least a portion of the target region and (ii) an associated DBR sequence of said DBR sequences; and

    c) estimating, using a computer, the number of initial target nucleic acid molecules that comprise said target region in said tagged sample based on;

    (i) a determination of the number of said different DBR sequences that are associated with said target region in the sequence reads; and

    (ii) a determination of the number of sequence reads that comprise each of the different DBR sequences that are associated with said target region.

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