ERROR CORRECTION IN AMPLIFICATION OF SAMPLES
First Claim
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1. A method comprising:
- stochastically barcoding a plurality of targets in a sample using a plurality of stochastic barcodes to generate stochastically barcoded targets, wherein each stochastic barcode comprises a molecular label and an identical sample label, and 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 the plurality of stochastic barcodes, wherein each of the one or more defined barcoded primers comprises a defined sample label and a defined molecular label, and wherein the defined barcoded primers are variants of the stochastic barcodes;
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
detecting the plurality of amplified stochastically barcoded targets and the plurality of amplified defined barcoded primers by detecting the molecular labels present on the plurality of stochastically barcoded targets and the defined molecular labels present on the plurality of amplified defined barcoded primers,wherein the number of molecular labels with different sequences associated with each of the plurality of targets in the plurality of amplified stochastically barcoded targets detected indicates a pre-correction number of the target,wherein the number of defined molecular labels in the plurality of amplified defined barcoded primers indicate an amplification noise, andwherein a difference between the number of molecular labels with different sequences associated with each of the plurality of targets and number of defined molecular labels with different sequences indicate the number of copies of the target.
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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.
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Citations
21 Claims
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1. A method comprising:
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stochastically barcoding a plurality of targets in a sample using a plurality of stochastic barcodes to generate stochastically barcoded targets, wherein each stochastic barcode comprises a molecular label and an identical sample label, and 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 the plurality of stochastic barcodes, wherein each of the one or more defined barcoded primers comprises a defined sample label and a defined molecular label, and wherein the defined barcoded primers are variants of the stochastic barcodes; 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 detecting the plurality of amplified stochastically barcoded targets and the plurality of amplified defined barcoded primers by detecting the molecular labels present on the plurality of stochastically barcoded targets and the defined molecular labels present on the plurality of amplified defined barcoded primers, wherein the number of molecular labels with different sequences associated with each of the plurality of targets in the plurality of amplified stochastically barcoded targets detected indicates a pre-correction number of the target, wherein the number of defined molecular labels in the plurality of amplified defined barcoded primers indicate an amplification noise, and wherein a difference between the number of molecular labels with different sequences associated with each of the plurality of targets and number of defined molecular labels with different sequences indicate the number of copies of the target. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)
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19. A method comprising:
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stochastically barcoding a plurality of targets in a sample using a plurality of stochastic barcodes, in the presence of one or more defined barcoded primers, to generate stochastically barcoded targets, wherein 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, wherein each of the one or more defined barcoded primers comprises a defined sample label and a defined molecular label, and wherein the defined barcoded primers are variants of the stochastic barcodes; 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 detecting the plurality of amplified stochastically barcoded targets and the plurality of amplified defined barcoded primers by detecting the molecular labels present on the plurality of stochastically barcoded targets and the defined molecular labels present on the plurality of amplified defined barcoded primers, wherein the number of molecular labels with different sequences associated with each of the plurality of targets in the plurality of amplified stochastically barcoded targets detected indicates a pre-correction number of the target, wherein the number of defined molecular labels in the plurality of amplified defined barcoded primers indicate an amplification noise, and wherein a difference between the number of molecular labels with different sequences associated with each of the plurality of targets and number of defined molecular labels with different sequences indicate the number of copies of the target.
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20. A method for determining amplification noise, comprising:
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reverse transcribing a plurality targets in a sample using oligonucleotides comprising reverse transcription primers to generate reverse transcribed targets; contacting one or more defined barcoded primers with the reverse transcribed targets, 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, wherein the number of defined molecular labels with different sequences in the plurality of defined barcoded amplified defined barcoded primers indicates an amplification noise.
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21. A method comprising:
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stochastically barcoding a plurality of targets in a sample in a plurality of partitions using a plurality of stochastic barcodes to generate stochastically barcoded targets, wherein for each partition, each stochastic barcode comprises a molecular label and an identical sample label, and 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 plurality of partitions to generate a pool of stochastically barcoded targets; contacting defined barcoded primers of one or more types of defined barcoded primers with the plurality of stochastic barcoded targets in the partitions, 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 detecting the plurality of amplified stochastically barcoded targets and the plurality of amplified defined barcoded primers; wherein the number of molecular labels with different sequences associated with each of the plurality of targets in the plurality of amplified stochastically barcoded targets detected indicates a pre-correction number of the target, wherein the number of defined molecular labels with different sequences in the plurality of amplified defined barcoded primers detected indicates an amplification noise, and wherein a difference between the number of molecular labels with different sequences associated with each of the plurality of targets and the number of defined molecular labels with different sequences indicate the number of copies of the target.
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Specification