Pan-genotypic probe-based enrichment to improve efficiency of Hepatitis B virus sequencing

Abstract

Hepatitis B Virus (HBV) genome sequencing can be used to provide more complete genetic information at the population and individual level to shed light on the limitations of current interventions, and inform new strategies for elimination. HBV sequencing is challenging due to the partially dsDNA genome, high diversity, low viral loads and presence of large amounts of host genetic material in clinical samples. Here we describe the design and use of a pan-genotypic panel of 74 HBV specific capture-probes and nuclease treatment in improving sequencing efficiency. We processed 20 plasma samples (viral loads 1.98 to 4.07 log10, genotypes A-E) and three positive controls (human total brain RNA and bacteriophage lambda DNA) in triplicate to compare DNAse vs. RNAse vs. no nuclease treatment. We prepared libraries using the Takara Bio SMARTer Stranded Total RNA-Seq Kit v3, split the library in two, enriching half with the custom-designed probe panel and xGen Hybridization and Wash Kit (IDT), the other half was not enriched. Both libraries were sequenced on the NovaSeq6000 platform with 2x150nt paired-end reads. Capture resulted in a 47,970 fold increase in the number of reads mapped to the HBV genome in the no nuclease arm (243 HBV reads per million reads sequenced in the capture pool vs. 5x10-3 reads per million in the no-capture pool). Out of 20 samples, only 1 without capture generated HBV reads (viral load 3.89 log10 IU/ml) vs. 19 samples with capture. HBV sequence yield was increased in the capture arm and resulted in 2.30 log10 (95% confidence interval 1.99 - 2.48 log10) increase in HBV reads (per million reads sequenced) per log10 increase in viral load. The proportion of HBV reads increased a median of 12 fold with RNAse treatment. We developed a targeted pan-genotypic sequencing method using a custom panel of biotinylated oligos that increases the sequencing efficacy of HBV. This method will allow us to gain a better insight into HBV diversity.

An enrichment protocol and analysis pipeline for long read sequencing of the hepatitis B virus transcriptome

“…cDNA quantity and quality were determined using a Nanodrop 2000 spectrophotometer (Thermo Scientific) and a Bioanalyzer 2100 (Agilent). A panel of 120 nucleotide (nt) biotinylated oligos targeting the entire HBV genome were designed using the xGen Lockdown platform (IDT) and have been described elsewhere [ 31 ]. Briefly, probes were designed to match the consensus of 4499 HBV whole genomes in the Hepatitis B Virus Database (hbvdb.lyon.inserm.fr/HBVdb, genotypes: A 506, B 1218, C 1447, D 823, E 254, F 197, G 28 and H 26).…”

“…Additional probes were synthesised for genotype G, which has a 36 base insertion in the core gene, and for genotype D that has a deletion of 33 bases in the pre-S1 region. Long capture probes of 120 nucleotides retain affinity for their target even with up to 20 % divergence [31,32]. Probes were incubated with the pooled cDNA at 65 °C for 4 h. The biotinylated primer-cDNA complexes were incubated with Streptavidin Dynabeads at 65 °C for 45 min followed by washing and elution (xGen Hybridization kit, IDT).…”

The N6-methyladenosine demethylase ALKBH5 regulates the hypoxic HBV transcriptome

“…Briefly, RNAs were prepared from normoxic or hypoxic HepG2-NTCP cells transfected with HBV HBV1.3 WT or m6-null constructs and 150ng of RNA reverse transcribed with barcoded sequencing primers. Oligonucleotide enrichment of HBV RNAs was performed as previously described [49]. Samples were sequenced using a Sequel II instrument to generate a PacBio ‘Hifi library’.…”

The N6-methyladenosine demethylase ALKBH5 regulates the hypoxic HBV transcriptome

“…Briefly, RNAs were prepared from normoxic or hypoxic HepG2-NTCP cells transfected with HBV HBV1.3 WT or m 6 A-null constructs and 150ng reverse transcribed with barcoded sequencing primers. Oligonucleotide enrichment of HBV RNAs was performed as previously described [73]. Samples were sequenced using a Sequel II instrument to generate a PacBio 'Hifi library'.…”