COMPOSITIONS AND METHOD FOR MEASURING AND CALIBRATING AMPLIFICATION BIAS IN MULTIPLEXED PCR REACTIONS

• US 20150203897A1
• Filed: 01/09/2015
• Published: 07/23/2015
• Est. Priority Date: 05/08/2012
• Status: Active Grant

• Current Assignee: Adaptive Biotechnologies Corp.
• Original Assignee: Adaptive Biotechnologies Corp.
• Inventors: ROBINS, HARLAN SAUL, CARLSON, CHRISTOPHER SCOTT, LIVINGSTON, ROBERT J., EMERSON, RYAN O., SHERWOOD, ANNA M.
• Granted Patent: US 9,371,558 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 2527/143 Concentration of primer or probe
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2545/101 with an internal standard/control
• CPC Class Codes: C12Q 2545/113 with an external standard/control, i.e. control reaction is separated from the test/target reaction
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: C12Q 2600/166 Oligonucleotides used as internal standards, controls or normalisation probes

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 N 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.

Claims

1. A composition for standardizing the amplification efficiency of an oligonucleotide primer set for amplifying rearranged nucleic acid sequences encoding one or more adaptive immune receptors in a biological sample of a mammalian subject, the composition comprising:

a plurality of synthetic template oligonucleotides, each synthetic template oligonucleotide having an oligonucleotide sequence comprising;

(a) a V sequence region comprising an oligonucleotide sequence comprising at least 20 contiguous nucleotides of an adaptive immune receptor variable (V) region encoding gene sequence, or the complement thereof, each V sequence region comprising a unique V-region oligonucleotide sequence;

(b) a J sequence region comprising an oligonucleotide sequence comprising at least 15 contiguous nucleotides of an adaptive immune receptor joining (J) region encoding gene sequence and flanking intron sequence, or the complement thereof, each J sequence region comprising a unique J-region oligonucleotide sequence;

(c) a U1 sequence comprising either nothing or a first sequencing platform-specific oligonucleotide sequence;

(d) a U2 sequence comprising either nothing or comprises a second sequencing platform-specific oligonucleotide sequence;

(e) at least one barcode sequence B1 comprising a sequence of 4-25 contiguous nucleotides that uniquely identifies, as a paired combination, (i) the unique V-region oligonucleotide sequence of (a) and (ii) the unique J-region oligonucleotide sequence of (b); and

(f) at least one random oligonucleotide sequence R comprising at least two contiguous nucleotides,

wherein the composition comprises at least one synthetic template oligonucleotide for each unique V-region oligonucleotide sequence and at least one synthetic template oligonucleotide for each unique J-region oligonucleotide sequence.

2. The composition of claim 1, wherein each synthetic template oligonucleotide in the plurality of synthetic template oligonucleotides is present in an equimolar amount.

3. The composition of claim 1, wherein each synthetic template oligonucleotide further comprises at least one additional barcode sequence.

4. The composition of claim 1, wherein each synthetic template oligonucleotide further comprises a restriction enzyme recognition site or other identifying site that comprises an oligonucleotide sequence that is absent from (a)-(e)

5. The composition of claim 1, wherein the random nucleotide sequence R is between 2 and 50 contiguous nucleotide in length.

6. The composition of claim 1, wherein the plurality of synthetic template oligonucleotides comprises a number of at least a or at least b unique oligonucleotide sequences, whichever is larger, wherein a is the number of unique adaptive immune receptor V region-encoding gene segments in the subject and b is the number of unique adaptive immune receptor J region-encoding gene segments in the subject.

7. The composition of claim 1, wherein a ranges from 1 to a number of maximum V gene segments in the mammalian genome of the subject and b ranges from 1 to a number of maximum J gene segments in the mammalian genome of the subject.

8. The composition of claim 1, wherein the plurality of synthetic template oligonucleotides comprises at least (a×

b) unique oligonucleotide sequences, where a is the number of unique adaptive immune receptor V region-encoding gene segments in the mammalian subject and b is the number of unique adaptive immune receptor J region-encoding gene segments in the mammalian subject, and the composition comprises at least one template oligonucleotide for every possible combination of a V region-encoding gene segment and a J region-encoding gene segment.

9. The composition of claim 1, wherein the adaptive immune receptor has a locus selected from the group consisting of TCRB, TCRG, TCRA, TCRD, IGH, IGK, and IGL.

10. The composition of claim 1, wherein the V sequence of (a) encodes a TCRB, TCRG, TCRA, TCRD, IGH, IGK, or IGL receptor V-region polypeptide and wherein the J sequence of (b) encodes a TCRB, TCRG, TCRA, TCRD, IGH, IGK, or IGL receptor J-region polypeptide.

11. The composition of claim 1, wherein V is an oligonucleotide sequence comprising at least 30 contiguous nucleotides and not more than 900 contiguous nucleotides of the adaptive immune receptor V region encoding gene sequence, or the complement thereof and wherein J is an oligonucleotide sequence comprising (i) at least 16 contiguous nucleotides and not more than 500 contiguous nucleotides of an adaptive immune receptor J region encoding gene sequence.

12. The composition of claim 1, wherein each synthetic template oligonucleotide is less than 1000 nucleotides in length.

13. The composition of claim 1, further comprising:

a set of oligonucleotide primers that is capable of amplifying rearranged nucleic acid molecules encoding one or more adaptive immune receptors comprising a plurality a′

of unique V-segment oligonucleotide primers and a plurality b′

of unique J-segment oligonucleotide primers and capable of amplifying said plurality of synthetic template oligonucleotides.

14. The composition of claim 13, wherein a′

ranges from 1 to a number of maximum V gene segments in the mammalian genome, and b′

ranges from 1 to a number of maximum number of J gene segments in the mammalian genome.

15. The composition of claim 13, wherein each V-segment oligonucleotide primer comprises a nucleotide sequence of at least 14 contiguous nucleotides that is complementary to at least one adaptive immune receptor V region-encoding gene segment and wherein each J-segment oligonucleotide primer comprises a nucleotide sequence of at least 15 contiguous nucleotides that is complementary to at least one adaptive immune receptor J region-encoding gene segment.

16. 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 the composition comprising said plurality of synthetic template oligonucleotides using the set of oligonucleotide primers of claim 13 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.

17. The method in claim 16, wherein said expected distribution is based on determining predetermined molar ratios of said plurality of synthetic template oligonucleotides comprising said composition.

18. The method of claim 17, wherein said predetermined molar ratios are equimolar.

19. The method in claim 16, wherein said expected distribution is determined based on frequencies of each unique synthetic template oligonucleotide, said frequencies determined based on quantifying the frequency of each unique random oligonucleotide sequence R.

20. The method of claim 16, wherein said expected distribution comprises a uniform amplification level for said plurality of synthetic template oligonucleotides amplified by said set of oligonucleotide primers.

21. The method of claim 16, further comprising adjusting the relative representation of the oligonucleotide primer member in the set of oligonucleotide amplification primers for each member of the set of oligonucleotide primers that exhibits non-uniform amplification potential relative to the expected distribution.

22. The method of claim 21, wherein adjusting comprises:

increasing the relative representation of the member in the set of oligonucleotide primers, thereby correcting non-uniform nucleic acid amplification potential among members of the set of oligonucleotide primers, or

decreasing the relative representation of the member in the set of oligonucleotide primers, thereby correcting non-uniform nucleic acid amplification potential among members of the set of oligonucleotide primers.

23. The method of claim 17, further comprising calculating a proportionately increased or decreased frequency of occurrence of the amplified template nucleic acid molecules for each member of the set of oligonucleotide amplification primers that exhibits non-uniform amplification potential relative to the expected distribution, thereby correcting for non-uniform nucleic acid amplification potential among members of the set of oligonucleotide primers.

24. A method for correcting for amplification bias in a single multiplex PCR amplification reaction to quantify rearranged nucleic acid molecules encoding a plurality of adaptive immune receptors in a biological sample obtained from a mammalian subject, comprising:

(a) contacting said sample with a composition comprising said plurality of synthetic template oligonucleotides and said set of oligonucleotide primers of claim 13 to generate a template-spiked sample;

(b) amplifying said template-spiked sample in a single multiplex PCR reaction using said set of oligonucleotide primers comprising said V-segment 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 from said subject;

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

(d) sequencing said plurality of amplified rearranged nucleic acid molecules from said subject encoding a plurality of adaptive immune receptors to determine (i) a rearranged nucleic acid molecule sequence and (ii) a frequency of occurrence of said rearranged nucleic acid molecule sequence for each unique rearranged nucleic acid molecule;

(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) optionally 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.