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

A method for correcting for amplification bias in a multiplex PCR amplification reaction to quantify rearranged nucleic acid molecules encoding one or a plurality of adaptive immune receptors in a biological sample that comprises rearranged nucleic acid molecules obtained from lymphoid cells of a mammalian subject, comprising:

• (a) contacting said sample with a composition comprising a plurality of synthetic template oligonucleotides and a set of oligonucleotide primers to generate a template-spiked sample, wherein said plurality of synthetic template oligonucleotides and said rearranged nucleic acid molecules comprise corresponding V and J region sequences,
• (b) amplifying said template-spiked sample in said multiplex PCR reaction using said set of oligonucleotide primers comprising V-segment oligonucleotide primers and J-segment oligonucleotide primers to obtain a plurality of amplified synthetic template oligonucleotides and a plurality of amplified rearranged nucleic acid molecules encoding a plurality of adaptive immune receptors;
• (c) sequencing said plurality of amplified synthetic template oligonucleotides to determine, for each unique template oligonucleotide comprising said plurality, (i) a synthetic template oligonucleotide sequence and (ii) a frequency of occurrence of said synthetic template oligonucleotide sequence;
• (d) sequencing said plurality of amplified rearranged nucleic acid molecules encoding one or a plurality of adaptive immune receptors, for each unique rearranged nucleic acid molecules encoding said plurality of adaptive immune receptors comprising said plurality, (i) a rearranged nucleic acid molecule sequence and (ii) a frequency of occurrence of said rearranged nucleic acid molecule sequence;
• (e) comparing said frequency of occurrence 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 primers;
• (f) generating a set of correction values for a set of synthetic template oligonucleotide sequences and rearranged nucleic acid molecule sequences amplified by said members of the set of oligonucleotide primers having said indicated non-uniform nucleic acid amplification potential, wherein said set of correction values corrects for amplification bias in said multiplex PCR reaction;
• and
• (g) applying said set of correction values to said frequency of occurrence of said rearranged nucleic acid molecule sequences to correct for amplification bias in said multiplex PCR reaction.