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AS004
Targeted long read sequencing reveals the comprehensive
architecture and expression patterns of integrated HBV DNA in
CHB liver biopsies
Ricardo Ramirez1, Nicholas Van Buuren1, Vithika Suri1, Henry Chan1,2,
Maria Buti1,3, Patrick Marcellin1,4, Hongmei Mo1, Anuj Gaggar1, Li Li1,
Becket Feierbach1. 1Gilead Sciences, Inc., Foster City, United States;
2Chinese University Of Hong Kong, Hong Kong; 3Vall d’Hebron University
Hospital, Barcelona, Spain; 4Hospital Beaujon AP-HP, Clichy, France
Email: [email protected]
Background and Aims: Hepatitis B virus (HBV) integration into the
host genome has been implicated in the development of hepatocellular
carcinoma and has been observed with PCR-based assays or
short read sequencing, but limitations of these methods have
prevented the full characterization of integration events. Here, we
use an HBV-targeted sequencing strategy to determine the full
architecture of HBV integrations in the liver of chronically-infected
HBV (CHB) patients. We define the full sequences of integrated HBV
DNA and compare these sequences with data from RNA-Seq to
determine productive vs non-productive integrations.
Method: 28 liver biopsies were obtained from CHB patients enrolled
in Gilead Clinical Trial GS-US-174-0149 with an even distribution of
HBeAg positive and negative samples, including five samples taken
96 weeks post-baseline. Genomic DNA was sheared to 7 kb
fragments, barcoded for multiplexing, and enriched for HBV using
the IDT xGen platform with a custom panel of biotinylated 120 bp
probes targeted to HBV genotypes A through H. Enriched DNA
libraries were sequenced using a Pacific Biosciences Sequel II (PacBio)
and compared to corresponding RNA-Seq.
Results: Target enrichment increased detection of HBV sequences,
and the PacBio platform produced high quality reads that were
several kilobases long, many of which contained the entire viral
integration sequence and thousands of base pairs of adjacent host
sequences within a single read. In contrast, short read whole genome sequencing data only detected virus-host junctions. We also
observed transcripts resulting from non-integrated HBV as identified
by the presence of 3.2 kb reads exclusive of host sequences. Host/HBV
chimeric reads were compared to RNA-Seq data from the same
biopsies to match transcripts with specific integration events. Several
partial integration events, not associated with HBV direct repeats
(DR1 and DR2), were identified and appeared transcriptionally silent
despite being inserted in or near host gene loci. Most transcribed
integrations contained HBV promoter sequences driving S transcript
expression and are associated with the HBV DR1 region.
Conclusion: Targeted long-read sequencing provides better accuracy
and deeper coverage, compared to whole genome sequencing. This
assay can elucidate full primary sequence of integrated HBV, and,
when paired with RNA-Seq data, can show the differences between
transcriptionally active and inactive events. |
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发表于 2020-8-23 13:04