title: Theoretical and experimental assessment of degenerate primer tagging in ultra-deep applications of next-generation sequencing.
abstract: Primer IDs (pIDs) are random oligonucleotide tags used in next-generation sequencing to identify sequences that originate from the same template. These tags are produced by degenerate primers during the reverse transcription of RNA molecules into cDNA. The use of pIDs helps to track the number of RNA molecules carried through amplification and sequencing, and allows resolution of inconsistencies between reads sharing a pID. Three potential issues complicate the above applications. First, multiple cDNAs may share a pID by chance; we found that while preventing any cDNAs from sharing a pID may be unfeasible, it is still practical to limit the number of these collisions. Secondly, a pID must be observed in at least three sequences to allow error correction; as such, pIDs observed only one or two times must be rejected. If the sequencing product contains copies from a high number of RT templates but produces few reads, our findings indicate that rejecting such pIDs will discard a great deal of data. Thirdly, the use of pIDs could influence amplification and sequencing. We examined the effects of several intrinsic and extrinsic factors on sequencing reads at both the individual and ensemble level.
authors.affiliation: BC Centre for Excellence in HIV/AIDS, Vancouver, BC, V6Z 1Y6, Canada rliang@cfenet.ubc.ca.
authors.forename: Richard H
authors.initials: RH
authors.lastname: Liang
authors.affiliation: BC Centre for Excellence in HIV/AIDS, Vancouver, BC, V6Z 1Y6, Canada.
authors.forename: Theresa
authors.initials: T
authors.lastname: Mo
authors.affiliation: BC Centre for Excellence in HIV/AIDS, Vancouver, BC, V6Z 1Y6, Canada.
authors.forename: Winnie
authors.initials: W
authors.lastname: Dong
authors.affiliation: BC Centre for Excellence in HIV/AIDS, Vancouver, BC, V6Z 1Y6, Canada.
authors.forename: Guinevere Q
authors.initials: GQ
authors.lastname: Lee
authors.affiliation: BC Centre for Excellence in HIV/AIDS, Vancouver, BC, V6Z 1Y6, Canada.
authors.forename: Luke C
authors.initials: LC
authors.lastname: Swenson
authors.affiliation: BC Centre for Excellence in HIV/AIDS, Vancouver, BC, V6Z 1Y6, Canada.
authors.forename: Rosemary M
authors.initials: RM
authors.lastname: McCloskey
authors.affiliation: BC Centre for Excellence in HIV/AIDS, Vancouver, BC, V6Z 1Y6, Canada.
authors.forename: Conan K
authors.initials: CK
authors.lastname: Woods
authors.affiliation: BC Centre for Excellence in HIV/AIDS, Vancouver, BC, V6Z 1Y6, Canada.
authors.forename: Chanson J
authors.initials: CJ
authors.lastname: Brumme
authors.affiliation: Section of Clinical Virology, Department of Medical Microbiology, Academic Medical Center, 1105AZ Amsterdam, The Netherlands.
authors.forename: Cynthia K Y
authors.initials: CK
authors.lastname: Ho
authors.affiliation: Section of Clinical Virology, Department of Medical Microbiology, Academic Medical Center, 1105AZ Amsterdam, The Netherlands.
authors.forename: Janke
authors.initials: J
authors.lastname: Schinkel
authors.affiliation: BC Centre for Excellence in HIV/AIDS, Vancouver, BC, V6Z 1Y6, Canada.
authors.forename: Jeffrey B
authors.initials: JB
authors.lastname: Joy
authors.affiliation: BC Centre for Excellence in HIV/AIDS, Vancouver, BC, V6Z 1Y6, Canada Department of Medicine, University of British Columbia, Vancouver, BC, V5Z 1M9, Canada.
authors.forename: P Richard
authors.initials: PR
authors.lastname: Harrigan
authors.affiliation: BC Centre for Excellence in HIV/AIDS, Vancouver, BC, V6Z 1Y6, Canada Department of Medicine, University of British Columbia, Vancouver, BC, V5Z 1M9, Canada.
authors.forename: Art F Y
authors.initials: AF
authors.lastname: Poon
mesh_terms: D017931:DNA Primers / Q000737:chemistry*; D018076:DNA, Complementary / Q000737:chemistry; D006678:HIV / Q000235:genetics; D016174:Hepacivirus / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000097:blood / Q000737:chemistry; D017423:Sequence Analysis, RNA
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D012367:RNA, Viral