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

  • US 9,150,905 B2
  • Filed: 05/08/2013
  • Issued: 10/06/2015
  • 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 that comprises rearranged nucleic acid molecules obtained from lymphoid cells of a mammalian subject, the method comprising:

  • (a) amplifying a plurality of synthetic template oligonucleotides comprising sequences of rearranged nucleic acid molecules encoding one or more adaptive immune receptors 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 said expected distribution is based on predetermined molar ratios of said plurality of synthetic template oligonucleotides comprising said composition, and 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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