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Hepatol Int. 2016 Jan;10(1):147-57. doi: 10.1007/s12072-015-9645-x. Epub 2015 Jul 25.

Aligning to the sample-specific reference sequence to optimize the accuracy of next-generation sequencing analysis for hepatitis B virus.

Liu WC1,2, Lin CP3, Cheng CP4, Ho CH5,6, Lan KL7, Cheng JH8, Yen CJ9,10, Cheng PN11, Wu IC12,13, Li IC14, Chang BC15, Tseng VS16, Chiu YC17,18, Chang TT19,20.

Author information

1
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.
2
Infectious Disease and Signaling Research Center, National Cheng Kung University, Tainan, Taiwan. graceliu8911@gmail.com.
3
Yourgene Bioscience, Taipei, Taiwan. darren@yourgene.com.tw.
4
Department of Computer Science and Information Engineering, National Cheng Kung University, Tainan, Taiwan. ccp0625@gmail.com.
5
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.
6
Infectious Disease and Signaling Research Center, National Cheng Kung University, Tainan, Taiwan. chenghsunho@gmail.com.
7
Department of Computer Science and Information Engineering, National Cheng Kung University, Tainan, Taiwan. lukelan305374@gmail.com.
8
Department of Computer Science and Information Engineering, National Cheng Kung University, Tainan, Taiwan. jihong@live.com.
9
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.
10
Infectious Disease and Signaling Research Center, National Cheng Kung University, Tainan, Taiwan. yencj@mail.ncku.edu.tw.
11
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.
12
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.
13
Infectious Disease and Signaling Research Center, National Cheng Kung University, Tainan, Taiwan. wichin@mail.ncku.edu.tw.
14
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.
15
Yourgene Bioscience, Taipei, Taiwan. bchang@yourgene.com.tw.
16
Department of Computer Science and Information Engineering, National Cheng Kung University, Tainan, Taiwan. tsengsm@mail.ncku.edu.tw.
17
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.
18
Infectious Disease and Signaling Research Center, National Cheng Kung University, Tainan, Taiwan. tannoy63352@gmail.com.
19
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.
20
Infectious Disease and Signaling Research Center, National Cheng Kung University, Tainan, Taiwan. ttchang@mail.ncku.edu.tw.

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.

KEYWORDS:

Alignment stage; Coverage; Divergence; Single nucleotide variants

PMID:
26208819
PMCID:
PMC4722079
DOI:
10.1007/s12072-015-9645-x
[Indexed for MEDLINE]
Free PMC Article

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