Methods using randomer-containing synthetic molecules

Patent metadata

Field	Value
Publication number	US-11248253-B2
Application number	US-202016864408-A
Country	US
Kind code	B2
Filing date	May 1, 2020
Priority date	Mar 5, 2014
Publication date	Feb 15, 2022
Grant date	Feb 15, 2022

Abstract

Methods are provided for correction of amplification bias and quantitation of adaptive immune cells in a sample using synthetic templates that include random oligonucleotide sequences.

First claim

The invention claimed is:

1. A method of correcting for amplification bias in a PCR reaction of a sample, the method comprising:
A) amplifying by multiplex PCR, sequencing, and quantifying output reads:
(i) biological template molecules comprising rearranged CDR3 oligonucleotide sequences from T cell receptor (TCR) loci from T cells or immunoglobulin (Ig) loci from B cells, each sequence comprising a TCR or IG V segment and a TCR or IG J segment, to obtain a total number of output biological sequence reads; and
(ii) synthetic template molecules each comprising one TCR or Ig V segment and one TCR or IG J segment, universal forward and/or reverse priming adaptor sequences, one or more barcodes that identify the template molecules as synthetic, an internal marker oligonucleotide sequence, and a random oligonucleotide sequence, wherein each random oligonucleotide sequence comprises a unique nucleotide sequence, and wherein each synthetic template molecule comprises a unique combination of a V segment and a J segment, to obtain a total number of output synthetic sequence reads;

B) clustering sequence reads by:
(i) extracting said sequence reads;
(ii) identifying whether a sequence read is a biological sequence read or a synthetic sequence read by:
(a) comparing the sequence reads against the known synthetic template sequences using a first metric in order to identify synthetic template sequences, while ignoring the portion of the sequence read expected to correspond to the random oligonucleotide sequence in synthetic sequences;
(b) comparing the remaining unmatched sequence reads against the known synthetic template sequences using a second metric, while ignoring the portion of the sequence read expected to correspond to the random oligonucleotide sequence in synthetic sequences;

(iii) clustering the synthetic sequence reads by collapsing sequence reads that match the same expected synthetic oligonucleotide sequence and share the same random oligonucleotide sequence;
(iv) assigning to each cluster of synthetic sequence reads, based on the expected synthetic oligonucleotide sequence to which they were matched, a consensus sequence comprising the expected sequence of the synthetic oligonucleotide sequence to which they were matched, including a V segment and a J segment; and
(v) determining a total number of observed synthetic sequence reads in each cluster;

C) calculating one or more normalization factors for the V segments and J segments in the synthetic sequence reads by:
(i) calculating a mean read count among all sequence clusters matched to each synthetic oligonucleotide sequence;
(ii) calculating an overall mean of the mean read counts for each unique V and J segment, among the synthetic oligonucleotide sequences containing a given V and any J or vice versa;
(iii) calculating a mean amplification bias by dividing the mean read count for each V segment and J segment calculated in step C (ii) by the overall mean V segment mean read counts or J segment mean read counts calculated in step C (ii) to arrive at an amplification factor for each V and J segment; and
(iv) producing the normalization factor for a given V or J segment by calculating the reciprocal of the mean amplification bias produced in step C (iii); and

D) multiplying the observed sequence read count of each given unique biological sequence by the normalization factor calculated in step C (iv) corresponding to the V segment present in that unique biological sequence and by the normalization factor calculated in step C (iv) corresponding to the J segment present in that unique biological sequence, thereby correcting for amplification bias in the multiplex PCR reaction of the sample.

2. The method of claim 1 , wherein the step of comparing the sequence reads against the known synthetic template sequences is performed with a Hamming metric.

3. The method of claim 1 , wherein the step of comparing the remaining unmatched sequence reads against the known synthetic template sequences is performed with a Levenshtein metric.

4. The method of claim 1 , wherein the sample is obtained from a mammalian subject.

5. The method of claim 1 , wherein the sample comprises a mixture of T cells and/or B cells, and cells that are not T cells or B cells.

6. The method of claim 1 , wherein said sample comprises human cells, mouse cells, or rat cells.

7. The method of claim 1 , wherein said sample comprises somatic tissue.

8. The method of claim 1 , wherein the sample is a tumor biopsy.

9. The method of claim 1 , wherein said sample is fresh tissue, frozen tissue, or fixed tissue.

10. The method of claim 1 , wherein the synthetic template molecules comprise the sequence of formula I:
5′-U1-B1-V-I-B2-N-J-B3-U2-3′, wherein

(i) V is an oligonucleotide sequence comprising at least 20 and not more than 1000 contiguous nucleotides of a TCR or Ig variable (V) region encoding gene sequence, or the complement thereof, and each synthetic template comprises a unique V-region oligonucleotide sequence;
(ii) J is an oligonucleotide sequence comprising at least 15 and not more than 600 contiguous nucleotides of a TCR or Ig joining (J) region encoding gene sequence, or the complement thereof, and each synthetic template comprises a unique V-region oligonucleotide sequence;
(iii) U1 comprises an oligonucleotide sequence that is selected from: (a) a first universal adaptor oligonucleotide sequence, and (b) a first sequencing platform oligonucleotide sequence that is linked to and positioned 5′ to a first universal adaptor oligonucleotide sequence;
(iv) U2 comprises an oligonucleotide sequence that is selected from: (a) a second universal adaptor oligonucleotide sequence, and (b) a second sequencing platform oligonucleotide sequence that is linked to and positioned 3′ to a second universal adaptor oligonucleotide sequence;
(v) I is an internal marker oligonucleotide sequence comprising at least 2 and not more than 100 nucleotides;
(vi) N is a random oligonucleotide sequence comprising at least 2 and not more than 100 nucleotides; and
(vii) B1, B2, and B3 each independently comprise either nothing or oligonucleotide barcode sequences of at least 2 and not more than 100 nucleotides that uniquely identify, as a pair combination, (a) said unique V region oligonucleotide sequences; and (b) said unique J region oligonucleotide sequences, wherein at least one of B1, B2, and B3 are present in each synthetic template.

11. The method of claim 10 , wherein N comprises at least 4 and not more than 15 nucleotides or wherein N comprises 8 nucleotides.

12. The method of claim 1 , wherein the amplification by multiplex PCR in step A is performed using a plurality of oligonucleotide primer sets comprising:
(a) a plurality of V segment oligonucleotide primers that are each independently capable of specifically hybridizing to at least one polynucleotide encoding a TCR or Ig V region polypeptide or to the complement thereof, wherein each V segment primer comprises a nucleotide sequence of at least 15 contiguous nucleotides that is complementary to at least one functional a TCR or Ig V region encoding gene segment and wherein said plurality of V segment primers specifically hybridize to substantially all functional TCR or Ig V region encoding gene segments that are present in the composition, and
(b) a plurality of J segment oligonucleotide primers that are each independently capable of specifically hybridizing to at least one polynucleotide encoding an TCR or Ig J region polypeptide or to the complement thereof, wherein each J segment primer comprises a nucleotide sequence of at least 15 contiguous nucleotides that is complementary to at least one fu

Assignees

• Adaptive Biotechnologies Corp

Inventors

• Sherwood Anna M
• Emerson Ryan O
• Robins Harlan S
• Rieder Mark J
• Parsons Joe

Classifications

• G16B30/00

  ICT specially adapted for sequence analysis involving nucleotides or amino acids · CPC title

• C07H21/00

  Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids · CPC title

• G01N33/566

  using specific carrier or receptor proteins as ligand binding reagents {where possible specific carrier or receptor proteins are classified with their target compounds} · CPC title

• C12Q1/68

  involving nucleic acids · CPC title

• C12N15/00

  Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor (mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00) · CPC title