Method of adding a DBR by primer extension
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 processing a genomic DNA sample, comprising:
(a) hybridizing a genomic sample comprising a population of initial target DNA molecules with a population of first primers that comprise: (i) a 3′ target-specific sequence that hybridizes to a sequence in said initial target DNA molecules, (ii) different degenerate base region (DBR) sequences, each of which is 5′ to said target-specific sequence, 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 (iii) a generic primer sequence that is 5′ to each of said DBR sequences, thereby producing duplexes comprising an initial target DNA molecule of said population of initial target DNA molecules and a primer of said first primers;
(b) subjecting the duplexes of step (a) to one, two or three rounds of primer extension to extend the population of first primers in said duplexes, thereby producing a mixture comprising tagged copies of said initial target DNA molecules, wherein each of said tagged copies comprises a DBR sequence of said DBR sequences and the generic primer sequence of said first population of primers;
(c) removing any of said population of first primers that have not been extended in step (b) from the mixture of step (b) or inactivating any of said population of first primers that have not been extended in step (b) in the mixture of step (b); and
(d) amplifying said tagged copies of said initial target DNA molecules after step c), thereby producing a plurality of different amplicons, wherein each of the different amplicons comprises multiple copies of the same polynucleotide, and wherein:
i) the amplifying step is done by polymerase chain reaction (PCR) using a primer pair that comprises a generic primer that hybridizes to the complement of the generic primer sequence of the first primers of step (a); and
ii) each of at least some of the different amplicons has a different DBR sequence of said DBR sequences.
2. The method of claim 1 , wherein step (b) comprises subjecting the duplexes of step (a) to one round of primer extension prior to step (c).
3. The method of claim 1 , wherein step (b) comprises subjecting the duplexes of step (a) to two rounds of primer extension prior to step (c).
4. The method of claim 1 , wherein said population of first primers have an affinity tag, and the removing or inactivating step (c) is done by immobilizing said population of first primers on a solid support.
5. The method of claim 1 , wherein the removing or inactivating step (c) comprises inactivating said population of first primers using an exonuclease.
6. The method of claim 1 , wherein said removing or inactivating step (c) comprises enzymatically modifying said population of first primers.
7. The method of claim 1 , wherein said DBR sequences comprises at least 2 nucleotide bases, wherein each of the at least 2 nucleotide bases are selected from: R, Y, S, W, K, M, B, D, H, V, N, and modified versions thereof.
8. The method of claim 7 , 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.
9. The method of claim 7 , wherein the DBR sequences comprise 10 or more-nucleotide bases, wherein each of the 10 or more nucleotide bases is selected from: R, Y, S, W, K, M, B, D, H, V, N, and modified versions thereof.
10. The method of claim 1 , wherein the initial target DNA molecules are enriched from an initial genomic sample prior to the hybridizing step (a).
11. The method of claim 1 , wherein the genomic sample comprises polynucleotides from a tumor.
12. The method of claim 1 , wherein the genomic sample comprises polynucleotides from a microorganism.
13. The method of claim 1 , wherein the genomic sample comprises polynucleotides from a virus.
14. The method of claim 1 , wherein the genomic sample comprises human genomic DNA.
15. The method of claim 1 , wherein the DBR sequences contain an error correcting code.
16. The method of claim 1 , wherein said genomic sample comprising a population of initial target DNA molecules is an amplification product.
17. The method of claim 1 , wherein said population of initial target DNA molecules are cDNA molecules made by reverse transcription of a viral genome.