title: Aligning to the sample-specific reference sequence to optimize the accuracy of next-generation sequencing analysis for hepatitis B virus.
abstract: BACKGROUND
  Hepatitis B virus (HBV) quasispecies are crucial in the pathogenesis of chronic liver disease. Next-generation sequencing (NGS) is powerful for identifying viral quasispecies. To improve mapping quality and single nucleotide variant (SNV) calling accuracy in the NGS analysis of HBV, we compared different mapping references, including the sample-specific reference sequence, same genotype sequences and different genotype sequences, according to the sample.
  
  
  METHODS
  Real Illumina HBV datasets from 86 patients, and simulated datasets from 158 HBV strains in the GenBank database, were used to assess mapping quality. SNV calling accuracy was evaluated using different mapping references to align Real Illumina datasets from a single HBV clone.
  
  
  RESULTS
  Using the sample-specific reference sequence as a mapping reference produced the largest number of mappable reads and coverages. With a different genotype mapping reference, the consensus sequence derived from the Real Illumina datasets of the single HBV clone showed 21 false SNV callings in polymerase and surface genes, the regions most divergent between the mapping reference and this HBV clone. A ~6 % coverage of most of these false SNVs was yielded even with a same genotype mapping reference, but none with the sample-specific reference sequence.
  
  
  CONCLUSIONS
  Using sample-specific reference sequences as a mapping reference in NGS analysis optimized mapping quality and the SNV calling accuracy for HBV quasispecies.
authors.affiliation: Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, 138 Sheng-Li Road, Tainan, 70403, Taiwan. graceliu8911@gmail.com.|Infectious Disease and Signaling Research Center, National Cheng Kung University, Tainan, Taiwan. graceliu8911@gmail.com.
authors.forename: Wen-Chun
authors.initials: WC
authors.lastname: Liu
authors.affiliation: Yourgene Bioscience, Taipei, Taiwan. darren@yourgene.com.tw.
authors.forename: Chih-Peng
authors.initials: CP
authors.lastname: Lin
authors.affiliation: Department of Computer Science and Information Engineering, National Cheng Kung University, Tainan, Taiwan. ccp0625@gmail.com.
authors.forename: Chun-Pei
authors.initials: CP
authors.lastname: Cheng
authors.affiliation: Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, 138 Sheng-Li Road, Tainan, 70403, Taiwan. chenghsunho@gmail.com.|Infectious Disease and Signaling Research Center, National Cheng Kung University, Tainan, Taiwan. chenghsunho@gmail.com.
authors.forename: Cheng-Hsun
authors.initials: CH
authors.lastname: Ho
authors.affiliation: Department of Computer Science and Information Engineering, National Cheng Kung University, Tainan, Taiwan. lukelan305374@gmail.com.
authors.forename: Kuo-Lun
authors.initials: KL
authors.lastname: Lan
authors.affiliation: Department of Computer Science and Information Engineering, National Cheng Kung University, Tainan, Taiwan. jihong@live.com.
authors.forename: Ji-Hong
authors.initials: JH
authors.lastname: Cheng
authors.affiliation: Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, 138 Sheng-Li Road, Tainan, 70403, Taiwan. yencj@mail.ncku.edu.tw.|Infectious Disease and Signaling Research Center, National Cheng Kung University, Tainan, Taiwan. yencj@mail.ncku.edu.tw.
authors.forename: Chia-Jui
authors.initials: CJ
authors.lastname: Yen
authors.affiliation: Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, 138 Sheng-Li Road, Tainan, 70403, Taiwan. pncheng@mail.ncku.edu.tw.
authors.forename: Pin-Nan
authors.initials: PN
authors.lastname: Cheng
authors.affiliation: Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, 138 Sheng-Li Road, Tainan, 70403, Taiwan. wichin@mail.ncku.edu.tw.|Infectious Disease and Signaling Research Center, National Cheng Kung University, Tainan, Taiwan. wichin@mail.ncku.edu.tw.
authors.forename: I-Chin
authors.initials: IC
authors.lastname: Wu
authors.affiliation: Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, 138 Sheng-Li Road, Tainan, 70403, Taiwan. ichenjellyli@gmail.com.
authors.forename: I-Chen
authors.initials: IC
authors.lastname: Li
authors.affiliation: Yourgene Bioscience, Taipei, Taiwan. bchang@yourgene.com.tw.
authors.forename: Bill Chia-Han
authors.initials: BC
authors.lastname: Chang
authors.affiliation: Department of Computer Science and Information Engineering, National Cheng Kung University, Tainan, Taiwan. tsengsm@mail.ncku.edu.tw.
authors.forename: Vincent S
authors.initials: VS
authors.lastname: Tseng
authors.affiliation: Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, 138 Sheng-Li Road, Tainan, 70403, Taiwan. tannoy63352@gmail.com.|Infectious Disease and Signaling Research Center, National Cheng Kung University, Tainan, Taiwan. tannoy63352@gmail.com.
authors.forename: Yen-Cheng
authors.initials: YC
authors.lastname: Chiu
authors.affiliation: Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, 138 Sheng-Li Road, Tainan, 70403, Taiwan. ttchang@mail.ncku.edu.tw.|Infectious Disease and Signaling Research Center, National Cheng Kung University, Tainan, Taiwan. ttchang@mail.ncku.edu.tw.
authors.forename: Ting-Tsung
authors.initials: TT
authors.lastname: Chang
mesh_terms: D000328:Adult; D000368:Aged; D001483:Base Sequence; D004279:DNA, Viral / Q000032:analysis; D005260:Female; D005838:Genotype; D006515:Hepatitis B virus / Q000145:classification / Q000235:genetics*; D019694:Hepatitis B, Chronic / Q000821:virology*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods* / Q000592:standards; D006801:Humans; D008297:Male; D008875:Middle Aged; D020641:Polymorphism, Single Nucleotide; D012016:Reference Values; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA / Q000379:methods* / Q000592:standards
chemical_list: D004279:DNA, Viral
keywords: Alignment stage; Coverage; Divergence; Single nucleotide variants