Method for accurately counting starting molecules
First Claim
1. A method for estimating the number of initial target nucleic acid molecules in a sample, comprising:
- a) amplifying a population of initial target nucleic acid molecules from a tagged sample thereby producing a population of amplified target DNA molecules, wherein the initial target nucleic acid molecules that comprise a target region are tagged with different degenerate base region (DBR) sequences, wherein said DBR sequences comprise at least one nucleotide base selected from;
R, Y, S, W, K, M, B, D, H, V, N and modified versions thereof and wherein the amplified target DNA molecules comprise said target region and an associated DBR sequence of said DBR sequences;
b) sequencing a plurality of the amplified target DNA molecules, thereby producing a plurality of sequence reads, wherein the sequencing step provides, for each of the amplified target DNA molecules that are sequenced, the nucleotide sequence of;
(i) at least a portion of the target region and (ii) an associated DBR sequence of said DBR sequences; and
c) estimating, using a computer, the number of initial target nucleic acid molecules that comprise said target region in said tagged sample based on;
(i) a determination of the number of said different DBR sequences that are associated with said target region in the sequence reads; and
(ii) a determination of the number of sequence reads that comprise each of the different DBR sequences that are associated with said target region.
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Accused Products
Abstract
Aspects of the present invention include methods and compositions for determining the number of individual polynucleotide molecules originating from the same genomic region of the same original sample that have been sequenced in a particular sequence analysis configuration or process. In these aspects of the invention, a degenerate base region (DBR) is attached to the starting polynucleotide molecules that are subsequently sequenced (e.g., after certain process steps are performed, e.g., amplification and/or enrichment). The number of different DBR sequences present in a sequencing run can be used to determine/estimate the number of different starting polynucleotides that have been sequenced. DBRs can be used to enhance numerous different nucleic acid sequence analysis applications, including allowing higher confidence allele call determinations in genotyping applications.
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Citations
22 Claims
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1. A method for estimating the number of initial target nucleic acid molecules in a sample, comprising:
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a) amplifying a population of initial target nucleic acid molecules from a tagged sample thereby producing a population of amplified target DNA molecules, wherein the initial target nucleic acid molecules that comprise a target region are tagged with different degenerate base region (DBR) sequences, wherein said DBR sequences comprise at least one nucleotide base selected from;
R, Y, S, W, K, M, B, D, H, V, N and modified versions thereof and wherein the amplified target DNA molecules comprise said target region and an associated DBR sequence of said DBR sequences;b) sequencing a plurality of the amplified target DNA molecules, thereby producing a plurality of sequence reads, wherein the sequencing step provides, for each of the amplified target DNA molecules that are sequenced, the nucleotide sequence of;
(i) at least a portion of the target region and (ii) an associated DBR sequence of said DBR sequences; andc) estimating, using a computer, the number of initial target nucleic acid molecules that comprise said target region in said tagged sample based on; (i) a determination of the number of said different DBR sequences that are associated with said target region in the sequence reads; and (ii) a determination of the number of sequence reads that comprise each of the different DBR sequences that are associated with said target region. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)
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Specification