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

Area of Science:

• Virology
• Genomics
• Bioinformatics

Background:

• Hepatitis B virus (HBV) quasispecies play a key role in chronic liver disease pathogenesis.
• Next-generation sequencing (NGS) is a vital tool for identifying viral quasispecies.
• Accurate analysis of HBV quasispecies is essential for understanding disease progression.

Purpose of the Study:

• To enhance mapping quality and single nucleotide variant (SNV) calling accuracy in NGS analysis of HBV.
• To compare the impact of different mapping references on HBV sequencing data.
• To identify the optimal reference sequence for accurate HBV quasispecies profiling.

Main Methods:

• Comparison of mapping references: sample-specific, same genotype, and different genotype sequences.
• Assessment of mapping quality using real Illumina HBV datasets (86 patients) and simulated datasets (158 strains).
• Evaluation of SNV calling accuracy using real Illumina datasets from a single HBV clone.

Main Results:

• Sample-specific reference sequences yielded the highest number of mappable reads and coverage.
• Using different genotype references resulted in 21 false SNV callings in polymerase and surface genes.
• False SNVs were detected with same genotype references, but not with sample-specific references.

Conclusions:

• Sample-specific reference sequences are optimal for NGS analysis of HBV quasispecies.
• This approach significantly improves mapping quality and SNV calling accuracy.
• Optimized NGS analysis aids in better understanding of HBV pathogenesis and disease management.