Compositions and method for measuring and calibrating amplification bias in multiplexed PCR reactions
First Claim
1. A method for quantifying a plurality of rearranged nucleic acid molecules encoding one or a plurality of adaptive immune receptors in a biological sample that comprises rearranged nucleic acid molecules from lymphoid cells of a mammalian subject, each adaptive immune receptor comprising a variable (V) region and a joining (J) region, the method comprising:
- (A) amplifying nucleic acid molecules in at least one multiplex polymerase chain reaction (PCR) that comprises;
(1) rearranged nucleic acid molecules from the biological sample that comprises lymphoid cells of the mammalian subject,(2) a composition, comprising a plurality of synthetic template oligonucleotides comprising sequences of rearranged nucleic acid molecules encoding one or more adaptive immune receptors and at least one unique oligonucleotide barcode sequence such that each synthetic template oligonucleotide has a unique oligonucleotide sequence and wherein a known number of each of the plurality of synthetic template oligonucleotides having a unique oligonucleotide sequence is present,(3) an oligonucleotide amplification primer set comprising;
a plurality of V-segment oligonucleotide primers and a plurality of J-segment oligonucleotide primers, wherein the primer set is capable of promoting amplification in said at least one multiplex polymerase chain reaction (PCR) of(i) substantially all synthetic template oligonucleotides in the composition to produce a multiplicity of amplified synthetic template oligonucleotides, said multiplicity of amplified synthetic template nucleic acid molecules being sufficient to quantify diversity of the synthetic template oligonucleotides in the composition, and(ii) substantially all rearranged nucleic acid molecules encoding adaptive immune receptors in the biological sample to produce a multiplicity of amplified rearranged nucleic acid molecules, said multiplicity of amplified rearranged nucleic acid molecules being sufficient to quantify diversity of the rearranged nucleic acid molecules in the nucleic acid from the biological sample,(B) quantitatively sequencing said amplified synthetic template oligonucleotides and said amplified rearranged nucleic acid molecules to quantify(i) a synthetic template product number of amplified template oligonucleotides which contain the at least one unique oligonucleotide barcode sequence, and(ii) a rearranged product number of amplified rearranged nucleic acid molecules which lack a unique oligonucleotide barcode sequence;
(C) calculating an amplification factor by dividing the synthetic template product number of (B)(i) by the known number of each of the plurality of synthetic template oligonucleotides having at least one unique barcode oligonucleotide sequence of (A)(2); and
(D) dividing the rearranged product number of (B)(ii) by the amplification factor calculated in (C) to quantify the number of unique adaptive immune receptor encoding rearranged nucleic acid molecules in the sample.
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Abstract
Compositions and methods are described for standardizing the DNA amplification efficiencies of a highly heterogeneous set of oligonucleotide primers as may typically be used to amplify a heterogeneous set of DNA templates that contains rearranged lymphoid cell DNA encoding T cell receptors (TCR) or immunoglobulins (IG). The presently disclosed embodiments are useful to overcome undesirable bias in the utilization of a subset of amplification primers, which leads to imprecision in multiplexed high throughput sequencing of amplification products to quantify unique TCR or Ig encoding genomes in a sample. Provided is a composition comprising a diverse plurality of template oligonucleotides in substantially equimolar amounts, for use as a calibration standard for amplification primer sets. Also provided are methods for identifying and correcting biased primer efficiency during amplification.
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Citations
23 Claims
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1. A method for quantifying a plurality of rearranged nucleic acid molecules encoding one or a plurality of adaptive immune receptors in a biological sample that comprises rearranged nucleic acid molecules from lymphoid cells of a mammalian subject, each adaptive immune receptor comprising a variable (V) region and a joining (J) region, the method comprising:
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(A) amplifying nucleic acid molecules in at least one multiplex polymerase chain reaction (PCR) that comprises; (1) rearranged nucleic acid molecules from the biological sample that comprises lymphoid cells of the mammalian subject, (2) a composition, comprising a plurality of synthetic template oligonucleotides comprising sequences of rearranged nucleic acid molecules encoding one or more adaptive immune receptors and at least one unique oligonucleotide barcode sequence such that each synthetic template oligonucleotide has a unique oligonucleotide sequence and wherein a known number of each of the plurality of synthetic template oligonucleotides having a unique oligonucleotide sequence is present, (3) an oligonucleotide amplification primer set comprising; a plurality of V-segment oligonucleotide primers and a plurality of J-segment oligonucleotide primers, wherein the primer set is capable of promoting amplification in said at least one multiplex polymerase chain reaction (PCR) of (i) substantially all synthetic template oligonucleotides in the composition to produce a multiplicity of amplified synthetic template oligonucleotides, said multiplicity of amplified synthetic template nucleic acid molecules being sufficient to quantify diversity of the synthetic template oligonucleotides in the composition, and (ii) substantially all rearranged nucleic acid molecules encoding adaptive immune receptors in the biological sample to produce a multiplicity of amplified rearranged nucleic acid molecules, said multiplicity of amplified rearranged nucleic acid molecules being sufficient to quantify diversity of the rearranged nucleic acid molecules in the nucleic acid from the biological sample, (B) quantitatively sequencing said amplified synthetic template oligonucleotides and said amplified rearranged nucleic acid molecules to quantify (i) a synthetic template product number of amplified template oligonucleotides which contain the at least one unique oligonucleotide barcode sequence, and (ii) a rearranged product number of amplified rearranged nucleic acid molecules which lack a unique oligonucleotide barcode sequence; (C) calculating an amplification factor by dividing the synthetic template product number of (B)(i) by the known number of each of the plurality of synthetic template oligonucleotides having at least one unique barcode oligonucleotide sequence of (A)(2); and (D) dividing the rearranged product number of (B)(ii) by the amplification factor calculated in (C) to quantify the number of unique adaptive immune receptor encoding rearranged nucleic acid molecules in the sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23)
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Specification