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

Abstract

Introduction: Serum 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.

Methods: Longitudinal 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.

Results: All 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.

Conclusions: This 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.

Keywords: hepatitis B virus; long-read sequencing; nanopore; pgRNA; serum HBV RNA; spliced RNA; transcript.

Figure 1

Bioinformatic methodologies for serum HBV RNA isoform identification. Basecalled, demultiplexed, and barcode binned data was used directly in the FLAIR workflow, while the RATTLE and Galaxy workflow arms used post-Pychopper and -Filtlong analyzed data. The Galaxy workflow uses Kraken 2, GraphMap, and length filtering within the online Galaxy infrastructure.

Figure 2

Transcript isoforms identified with RATTLE, FLAIR, and using the Galaxy workflow. Transcript coordinates are based on GenBank sequence AB368296 and exclude the poly(A) tail. Dotted lines indicate spliced sequence and arrows indicate the 5′ and 3′ ends of the sequence and the location of transcript truncation, assumed to be an artifact of sequencing. Quantities of each transcript isoform are not reflected, although proportions of each transcript type are shown in Table 3. For samples having up to three 3′ truncated variants detected, the 5′ and 3′ ends are given consecutively in order of length. For samples having more than three 3′ truncated variants detected, the 5′ and 3′ ends are denoted in italics for multiple variants and the range of variant lengths is shown by a diagonal line bar (as with the Galaxy workflow), with the shortest and longest transcript coordinates shown. Only HBx mRNAs containing all 3 sequence markers when using the Galaxy workflow are reflected. A linearized HBV genome showing reading frames (individual rectangles prefaced by the name of the open reading frame) and transcription start sites (bent arrows on the upper line denoting the complete genome) used for reference is shown at the top of the figure. Spliced variants found in poly(A) fractions were included here but excluded from further analyses.

Figure 3

Serum HBV RNA isoforms identified in pregnant individuals sampled longitudinally. Five transcript types (full-length and putative pgRNA, HBx mRNA, spliced variants, 3′ truncated variants) were analyzed at two time points from 3 pregnant individuals; time point #1 was during pregnancy (post 22-weeks) and time point #2 was post-partum (approximately 7 months between time points).