ERROR CORRECTION IN AMPLIFICATION OF SAMPLES

• US 20170342465A1
• Filed: 05/30/2017
• Published: 11/30/2017
• Est. Priority Date: 05/31/2016
• Status: Active Grant

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Cellular Research, Inc. (Becton, Dickinson & Co)
• Inventors: Shum, Eleen
• Granted Patent: US 10,202,641 B2
• Time in Patent Office: Days
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide

Abstract

Disclosed herein are methods and systems for correcting errors in sample amplification, including the errors occurred in determining the number of targets in samples. In some embodiments, the method comprises: stochastically barcoding a plurality of targets in the samples using oligonucleotides comprising stochastic barcodes to generate stochastically barcoded targets; contacting one or more defined barcoded primers with each of the one or more samples; and determining an amplification noise.

Claims

1. A method for determining the number of targets in one or more samples, comprising:

stochastically barcoding a plurality of targets in each of one or more samples using a plurality of oligonucleotides comprising stochastic barcodes to generate stochastically barcoded targets, wherein for each sample, each stochastic barcode comprises a molecular label and an identical sample label, wherein the molecular labels of at least two stochastic barcodes differ from one another by at least one nucleotide;

contacting one or more defined barcoded primers with each of the one or more samples, wherein each of the one or more defined barcoded primers comprises a defined sample label and a defined molecular label, and wherein the defined sample labels are variants of the sample labels;

amplifying the stochastically barcoded targets and the one or more defined barcoded primers to generate a plurality of amplified stochastically barcoded targets and a plurality of amplified defined barcoded primers; and

estimating the number of each of the plurality of targets, wherein estimating the number of each of the plurality of targets comprises;

determining a pre-correction number of each of the plurality of targets using the molecular label;

determining an amplification noise by determining the number of defined molecular labels with different sequences in the plurality of amplified defined barcoded primers; and

removing the amplification noise from the pre-correction number of each of the plurality of targets to generate the estimated number of each of the plurality of targets.

2. The method of claim 1, wherein contacting the one or more defined barcoded primers with each of the one or more samples comprises introducing the one or more defined barcoded primers at the same concentration of the plurality of oligonucleotides comprising the stochastic barcodes.

3. The method of claim 1, wherein the defined molecular label is 5-20 nucleotides in length, or wherein the defined molecular label and the molecular label have the same length.

5. The method of claim 1, wherein some of the molecular labels of the stochastic barcodes and some of the defined molecular labels of the one or more defined barcoded primers have the same sequence.

6. The method of claim 1, wherein the defined sample label is 5-20 nucleotides in length.

7. The method of claim 1, wherein the one or more defined barcoded primers comprise one or more types of defined barcoded primers.

8. The method of claim 7, wherein the defined molecular labels of one type of defined barcoded primers differ from one another by at least one nucleotide.

9. The method of claim 7,

wherein some defined molecular labels of different types of defined barcoded primers have the same sequence,.

wherein different types of defined barcoded primers have the same length,

wherein the lengths of different types of defined barcoded primers differ by at most 10 nucleotides, or

wherein the defined sample labels of different types of defined barcoded primers have different sequences.

13. The method of claim 1, wherein the sample label and the defined sample label have the same length.

14. The method of claim 1, wherein the Hamming distance between the sample labels of the stochastic barcodes and the defined sample labels of the defined barcoded primers is at least 2.

16. The method of claim 7, wherein the stochastic barcodes and the one or more types of defined barcoded primers have the same length.

18. The method of claim 1, further comprising removing at least 50% of the oligonucleotides comprising stochastic barcodes not incorporated into the stochastically barcoded targets and the one or more defined barcoded primers from each of the one or more samples.

19. The method of claim 18,

wherein less than 10% of the unincorporated oligonucleotides comprising the stochastic barcodes are not removed from each of the one or more samples,

wherein the percentage of the one or more defined barcoded primers not removed from each of the one or more samples is substantially the same as the percentage of the plurality of oligonucleotides comprising the stochastic barcodes not removed from each of the one or more samples, or

wherein the percentage of the one or more defined barcoded primers not removed from each of the one or more samples is within 10% of the percentage of the plurality of oligonucleotides comprising the stochastic barcodes not removed from each of the one or more samples.

23. The method of claim 1,

wherein determining the pre-correction number of each of the plurality of targets using the molecular label comprises:

determining sequences of molecular labels of the amplified stochastically barcoded targets; and

counting the number of the molecular labels with different sequences, and

wherein determining the number of defined molecular labels with different sequences in the plurality of amplified defined barcoded primers comprises;

determining sequences of the defined molecular labels of the amplified defined barcoded primers; and

counting the number of defined molecular labels with different sequences.

29. The method of claim 1, wherein determining the amplification noise comprises one or more of:

determining the number of defined molecular labels in the plurality of amplified defined barcoded primers with different sequences for each type of amplified defined barcoded primers,

determining the average number of defined molecular labels in the plurality of amplified defined barcoded primers with different sequences for different types of amplified defined barcoded primers, and

determining the maximum number of defined molecular labels in the plurality of amplified stochastically barcoded targets and the plurality of amplified defined barcoded primers with different sequences for different types of amplified defined barcoded primers.

32. The method of claim 1, wherein the amplification noise comprises noise caused by PCR crossover.

42. The method of claim 1, wherein the one or more samples comprise cells, and wherein at least one of the one or more samples comprises a single cell, the method comprising lysing the cells.

4. (canceled)

10-12. -12. (canceled)

15. (canceled)

17. (canceled)

20-22. -22. (canceled)

24-28. -28. (canceled)

30-31. -31. (canceled)

33-41. -41. (canceled)

43-49. -49. (canceled)

50. A method for determining amplification noise, comprising:

reverse transcribing a plurality targets in each of one or more samples using oligonucleotides comprising reverse transcription primers to generate reverse transcribed targets;

contacting one or more defined barcoded primers with each of the one or more samples, wherein each of the one or more defined barcoded primers comprises a defined molecular label;

amplifying the reverse transcribed targets and the one or more defined barcoded primers to generate a plurality of amplified reverse transcribed targets and a plurality of amplified defined barcoded primers; and

determining an amplification noise by determining the number of defined molecular labels with different sequences in the plurality of defined barcoded primers.

51-98. -98. (canceled)

99. A method for determining the number of targets in microwells of a microwell array, comprising:

stochastically barcoding a plurality of targets in each of one or more samples in microwells of a microwell array using a plurality of oligonucleotides comprising stochastic barcodes to generate stochastically barcoded targets, wherein for each well, each stochastic barcode comprises a molecular label and an identical sample label, wherein the molecular labels of at least two stochastic barcodes differ from one another by at least one nucleotide;

pooling the stochastically barcoded targets from the microwells of the microwell array to generate a pool of stochastically barcoded targets;

contacting defined barcoded primers of one or more types of defined barcoded primers with each of the one or more samples, wherein each defined barcoded primer comprises a defined sample label and a defined molecular label, wherein the defined sample labels of defined barcoded primers of the same type of defined barcoded primers have the same sequence, wherein the defined sample labels of different types of defined barcoded primers have different sequences, wherein the sample labels of the stochastic barcodes and the defined sample labels are variants of the sample label, and wherein the Hamming distance between the sample labels of the stochastic barcodes and the defined sample labels of the defined barcoded primers is at least 2;

amplifying the stochastically barcoded targets and the one or more defined barcoded primers to generate a plurality of amplified stochastically barcoded targets and a plurality of amplified defined barcoded primers; and

estimating the number of each of the plurality of targets, wherein estimating the number of each of the plurality of targets comprises;

determining a pre-correction number of each of the plurality of targets using the molecular label;

determining an amplification noise by determining the number of defined molecular labels with different sequences in the plurality of amplified defined barcoded primers; and

removing the amplification noise from the pre-correction number of each of the plurality of targets to generate the estimated number of each of the plurality of targets.

100-142. -142. (canceled)

143. A kit comprising:

a plurality of oligonucleotides comprising stochastic barcodes, wherein each stochastic barcode comprises a sample label and a molecular label, wherein the sample labels of at least two stochastic barcodes have different sequences, and wherein the molecular labels of stochastic barcodes with sample labels of the same sequence differ from one another by at least one nucleotide;

defined barcoded primers, wherein each defined barcoded primer comprises a defined sample label and a defined molecular label, wherein the defined sample labels of defined barcoded primers are variants of the sample labels of the stochastic barcodes, and wherein the Hamming distance between the sample label and the defined sample label is at least 2; and

instructions for using the plurality oligonucleotides comprising the plurality of oligonucleotides and the defined barcoded primers.

144-163. -163. (canceled)