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Compositions and method for measuring and calibrating amplification bias in multiplexed PCR reactions

  • US 9,371,558 B2
  • Filed: 01/09/2015
  • Issued: 06/21/2016
  • Est. Priority Date: 05/08/2012
  • Status: Active Grant
First Claim
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1. A method for determining non-uniform nucleic acid amplification potential among members of a set of oligonucleotide primers that is capable of amplifying rearranged nucleic acid molecules encoding one or more adaptive immune receptors in a biological sample from a mammalian subject, the method comprising:

  • (a) amplifying a composition comprising a plurality of synthetic template oligonucleotides comprising sequences of rearranged nucleic acid molecules encoding one or more adaptive immune receptors and one or more additional unique random polynucleotide sequences using a set of oligonucleotide primers in a single multiplex PCR reaction to obtain a plurality of amplified synthetic template oligonucleotides;

    (b) sequencing said plurality of amplified synthetic template oligonucleotides to determine, for each unique synthetic template oligonucleotide comprising said plurality,(i) a synthetic template oligonucleotide sequence and(ii) a frequency of occurrence of said synthetic template oligonucleotide sequence; and

    (c) comparing a frequency of occurrence of each of said synthetic template oligonucleotide sequences to an expected distribution, wherein a deviation between said frequency of occurrence of said synthetic template oligonucleotide sequences and said expected distribution indicates a non-uniform nucleic acid amplification potential among members of the set of oligonucleotide amplification primers.

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