Increased confidence of allele calls with molecular counting
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.
1. A method for assessing the presence of a genetic variation in a tagged viral sample, comprising:
(a) amplifying a population of target DNA molecules from the tagged viral sample, thereby producing a population of amplified target DNA molecules, wherein at least some of the target DNA molecules are tagged with different degenerate base region (DBR) sequences, wherein said DBR sequences comprises at least one nucleotide base selected from: R, V, S, W, K, M, B, D, H, V, N and modified versions thereof and wherein each of the amplified target DNA molecules comprises an associated DBR sequence of said DBR sequences;
(b) sequencing at least some of the amplified target DNA molecules of step (a), thereby producing a plurality of sequence reads, wherein the sequencing step provides, for each of the amplified target DNA molecules that are sequenced: (i) the nucleotide sequence of a target DNA molecule in the amplified target DNA molecules and (ii) the nucleotide sequence of an associated DBR sequence of said DBR sequence;
(c) examining the sequence reads of step (b), thereby identifying a potential genetic variation; and
(d) assessing, using a computer, the presence of the genetic variation in said tagged viral sample, based on:
(i) a determination of the number of said different DBR sequences that are associated with said genetic variation; and
(ii) a determination of the number of said sequence reads that comprise each of the different DBR sequences that are associated with said genetic variation.
2. The method of claim 1 , wherein the method comprises performing steps (a) to (d) on a plurality of different samples collected over time from the same subject.
3. The method of claim 1 , wherein the target DNA molecules are cDNA molecules.
4. The method of claim 1 , wherein said population of target DNA molecules is made by primer extension of a set of primers that comprises said DBR sequences and using a viral nucleic acid sample as a template.
5. The method of claim 1 , wherein said population of target DNA molecules is made by ligating a set of adaptors that comprise said DBR sequences to a viral nucleic acid sample.
6. The method of claim 1 , wherein the method comprises, prior to said amplifying step (a), enriching for said population of DNA molecules from the tagged viral sample.
7. The method of claim 1 , wherein the DBR sequences comprise 3 to 10 nucleotide bases, wherein each of the 3 to 10 nucleotide bases is selected from: R, Y, S, W, K, M, B, D, H, V, N, and modified versions thereof.
8. The method of claim 1 , wherein said DBR sequences comprise an error-correcting code.
9. The method of claim 1 , wherein the target DNA molecules are further tagged with a unique multiplex identifier (MID) sequence that identifies the source of a nucleic acid molecule.
10. The method of claim 1 , wherein said tagged viral sample is a pooled sample comprising nucleic acid molecules from several different sources, wherein each of said sources is associated with a unique multiplex identifier sequence that identifies the source of each of the nucleic acid molecules.
11. The method of claim 1 , wherein the sequencing step is done using a next-generation sequencing platform.
12. The method of claim 1 , wherein the amplifying step is done by polymerase chain reaction.
13. The method of claim 1 , wherein said tagged viral sample is an amplification product.
14. The method of claim 1 , wherein step (d) further comprises performing a maximum likelihood analysis.
15. The method of claim 1 , wherein the tagged viral sample contains different viral polynucleotide species, and the levels of the different viral polynucleotide species associated with the genetic variation are assessed.
16. The method of claim 1 , wherein the amplifying step comprises amplifying a region of interest from a population of target DNA molecules from the tagged viral sample, and step (c) further comprises examining the sequence reads of step b), thereby identifying a potential genetic variation in the region of interest.
17. A method for assessing the presence of a genetic variation in a tagged microbial sample, comprising:
(a) amplifying a population of target DNA molecules from the tagged microbial sample, thereby producing a population of amplified target DNA molecules, wherein at least some of the target DNA molecules are tagged with different degenerate base region (DBR) sequences, wherein said DBR sequences comprises at least one nucleotide base selected from: R, V, S, W, K, M, B, I), H, V, N and modified versions thereof and wherein each of the amplified target DNA molecules comprises an associated DBR sequence of said DBR sequences;
(b) sequencing at least some of the amplified target DNA molecules of step (a), thereby producing a plurality of sequence reads, wherein the sequencing step provides, for each of the amplified target DNA molecules that are sequenced: (i) the nucleotide sequence of a target DNA molecule in the amplified target DNA molecules and (ii) the nucleotide sequence of an associated DBR sequence of said DBR sequences;
(c) examining the sequence reads of step (b), thereby identifying a potential genetic variation; and
(d) assessing, using a computer, the presence of the genetic variation in said tagged microbial sample, based on:
(i) a determination of the number of said different DBR sequences that are associated with said genetic variation; and
(ii) a determination of the number of said sequence reads that comprise each of the different DBR sequences that are associated with said genetic variation.
18. The method of claim 17 , wherein the region of the population of target DNA molecules amplified in step (a) is antibiotic resistance gene, a metabolic enzyme gene or a gene encoding a ribosomal RNA.
19. The method of claim 17 , wherein the tagged microbial sample comprises DNA from different microbes and the method further comprises determining genetic diversities of the different microbes.
20. The method of claim 17 , wherein the method comprises performing steps (a) to (d) on a plurality of different samples collected over time from the same subject.
21. The method of claim 1 , wherein the amplifying step comprises amplifying a region of interest from a population of target DNA molecules from the tagged microbial sample, and step (c) further comprises examining the sequence reads of step b), thereby identifying a potential genetic variation in the region of interest.