Data_Sheet_1_Hepatitis B virus serum RNA transcript isoform composition and proportion in chronic hepatitis B patients by nanopore long-read sequencing.zip

IntroductionSerum hepatitis B virus (HBV) RNA is a promising new biomarker to manage and predict clinical outcomes of chronic hepatitis B (CHB) infection. However, the HBV serum transcriptome within encapsidated particles, which is the biomarker analyte measured in serum, remains poorly characterized. This study aimed to evaluate serum HBV RNA transcript composition and proportionality by PCR-cDNA nanopore sequencing of samples from CHB patients having varied HBV genotype (gt, A to F) and HBeAg status.
MethodsLongitudinal specimens from 3 individuals during and following pregnancy (approximately 7 months between time points) were also investigated. HBV RNA extracted from 16 serum samples obtained from 13 patients (73.3% female, 84.6% Asian) was sequenced and serum HBV RNA isoform detection and quantification were performed using three bioinformatic workflows; FLAIR, RATTLE, and a GraphMap-based workflow within the Galaxy application. A spike-in RNA variant (SIRV) control mix was used to assess run quality and coverage. The proportionality of transcript isoforms was based on total HBV reads determined by each workflow.
ResultsAll chosen isoform detection workflows showed high agreement in transcript proportionality and composition for most samples. HBV pregenomic RNA (pgRNA) was the most frequently observed transcript isoform (93.8% of patient samples), while other detected transcripts included pgRNA spliced variants, 3′ truncated variants and HBx mRNA, depending on the isoform detection method. Spliced variants of pgRNA were primarily observed in HBV gtB, C, E, or F-infected patients, with the Sp1 spliced variant detected most frequently. Twelve other pgRNA spliced variant transcripts were identified, including 3 previously unidentified transcripts, although spliced isoform identification was very dependent on the workflow used to analyze sequence data. Longitudinal sampling among pregnant and post-partum antiviral-treated individuals showed increasing proportions of 3′ truncated pgRNA variants over time.
ConclusionsThis study demonstrated long-read sequencing as a promising tool for the characterization of the serum HBV transcriptome. However, further studies are needed to better understand how serum HBV RNA isoform type and proportion are linked to CHB disease progression and antiviral treatment response.

引言
血清乙型肝炎病毒（HBV）RNA是用于管理和预测慢性乙型肝炎（CHB）感染临床结局的极具潜力的新型生物标志物。然而，作为血清中检测的生物标志物分析物的衣壳化颗粒内的HBV血清转录组，其特征仍未得到充分阐明。本研究旨在通过对不同乙型肝炎病毒基因型（gt，A至F）及乙型肝炎e抗原（HBeAg）状态的慢性乙型肝炎患者样本进行PCR-cDNA纳米孔测序，评估血清HBV RNA的转录组成及比例关系。
方法
本研究同时对3名个体在妊娠期间及产后（时间点间隔约7个月）的纵向标本进行了分析。从13名患者（其中女性占73.3%，亚裔占84.6%）的16份血清样本中提取HBV RNA并进行测序，随后采用三种生物信息学流程——FLAIR、RATTLE以及Galaxy应用内基于GraphMap的流程——完成血清HBV RNA异构体的检测与定量。使用外参RNA变异体（SIRV）对照混合物评估测序运行质量及覆盖度。转录异构体的比例关系基于各流程测定的总HBV读段数确定。
结果
所有选定的异构体检测流程在多数样本的转录本比例及组成上均表现出高度一致性。HBV前基因组RNA（pgRNA）是最常检测到的转录异构体（占患者样本的93.8%），其余检测到的转录本包括pgRNA剪接变体、3'端截短变体及HBx信使RNA（HBx mRNA），具体检出类型取决于异构体检测方法。pgRNA的剪接变体主要见于感染HBV gtB、C、E或F的患者，其中Sp1剪接变体检出频率最高。本研究共鉴定出12种其他pgRNA剪接变异转录本，其中包括3种此前未被报道的转录本，不过剪接异构体的鉴定高度依赖用于分析测序数据的流程。对接受抗病毒治疗的妊娠及产后个体的纵向采样分析显示，3'端截短pgRNA变体的比例随时间推移逐渐升高。
结论
本研究证实长读长测序是用于解析血清HBV转录组的极具潜力的工具。然而，仍需开展进一步研究以更好地阐明血清HBV RNA异构体的类型与比例如何与慢性乙型肝炎疾病进展及抗病毒治疗应答相关联。