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

Abstract

BackgroundHepatitis 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.MethodsReal 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.ResultsUsing 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.ConclusionsUsing sample-specific reference sequences as a mapping reference in NGS analysis optimized mapping quality and the SNV calling accuracy for HBV quasispecies.Electronic supplementary materialThe online version of this article (doi:10.1007/s12072-015-9645-x) contains supplementary material, which is available to authorized users.

Breed-specific Reference Sequence Optimizes Mapping Accuracy of NGS Analyses for Pigs

“…Moreover, different reference sequences also might mean different performances on mapping. Liu et al found when the sample-speci c sequence, a sequence with the same genotype sequence as the sample, was used as the mapping reference in NGS analyses of HBV(Hepatitis B Virus), the mapping accuracy and variation calling were optimized, compared to when the other four commonly used HBV genome sequences from GenBank database were respectively used as the mapping references [15].The present study provides an important recommendation for the selection of reference sequences for homology analysis. We compared three references ( the mitogenome alignment showed the brilliant performance than the other two references.…”

“…Optimized highly similar sequences have always been an important selection of reference sequences during the homologous sequences alignment to assist in piecing reads together. In order to accurately construct a genome sequence from NGS data, many studies turn to exploring the optimal sequencing methods and alignment strategies [14][15][16][17][18][19][20]. The de novo and reference-based approaches were used to assemble the complete mitogenome of Clarias batrachus from NGS data, respectively, which resulted in different consensus sequences in length [14].…”

“…With NGS data, the complete mitogenomes of many mammals have been obtained, including pigs [3][4][5][6], chickens [7][8][9] and cattle [10][11][12][13]. It is reported that reference sequences affected the accuracy in genome mapping [14][15][16]. Despite the existence of relevant studies, there are few studies on the effect of reference sequences on genome mapping and the quality of NGS in mammals or agricultural animals.…”

The Study of Hepatitis B Virus Using Bioinformatics

“…NGS or ultra-deep sequencing generates large volumes of data, which can only be analyzed using bioinformatics tools and provides large coverage that can detect minor quasispecies populations of HBV [46][47][48][49][50][51] that may be important in understanding HBV pathogenicity and response to treatment. In order to minimize the number of artifactual calls of single-nucleotide variations in NGS, it is important that the correct reference sequences are used [51,52].…”

Applications of next-generation sequencing analysis for the detection of hepatocellular carcinoma-associated hepatitis B virus mutations

“…A sample-specific reference sequence is the consensus sequence obtained from the NGS reads of each sample through alignment with its same genotype mapping reference. In our demonstrations, we found that using this type of reference sequence as the mapping reference has the best mapping quality and the highest single nucleotide variant (SNV) calling accuracy, as compared with using the compatible genotype sequence [ 66 ]. The percentage of false SNV calls increased significantly from 0.09% using a sample-specific reference sequence to 28.95% using an incompatible genotype reference (Fig.…”