{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T17:42:41Z","timestamp":1790790161657,"version":"4.1.0"},"reference-count":74,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2023,8,14]],"date-time":"2023-08-14T00:00:00Z","timestamp":1691971200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:sec><jats:title>Introduction<\/jats:title><jats:p>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.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>Longitudinal specimens from 3 individuals during and following pregnancy (approximately 7\u2009months 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.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>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\u2032 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\u2032 truncated pgRNA variants over time.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusions<\/jats:title><jats:p>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.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fmicb.2023.1233178","type":"journal-article","created":{"date-parts":[[2023,8,14]],"date-time":"2023-08-14T16:25:53Z","timestamp":1692030353000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":15,"title":["Hepatitis B virus serum RNA transcript isoform composition and proportion in chronic hepatitis B patients by nanopore long-read sequencing"],"prefix":"10.3389","volume":"14","author":[{"given":"Alicia","family":"Vachon","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Grace E.","family":"Seo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nishi H.","family":"Patel","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carla S.","family":"Coffin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eric","family":"Marinier","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eduardo","family":"Eyras","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carla","family":"Osiowy","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2023,8,14]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"523","DOI":"10.1007\/s10142-020-00732-1","article-title":"UNAGI: an automated pipeline for nanopore full-length cDNA sequencing uncovers novel transcripts and isoforms in yeast","volume":"20","author":"Al Kadi","year":"2020","journal-title":"Funct. 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Virol."},{"key":"ref5","doi-asserted-by":"publisher","first-page":"798","DOI":"10.1128\/JVI.00798-18","article-title":"Extracellular hepatitis B virus RNAs are heterogeneous in length and circulate as capsid-antibody complexes in addition to virions in chronic hepatitis B patients","volume":"92","author":"Bai","year":"2018","journal-title":"J. Virol."},{"key":"ref6","doi-asserted-by":"publisher","first-page":"15989","DOI":"10.1038\/s41598-017-16262-z","article-title":"Long-read sequencing of human cytomegalovirus transcriptome reveals RNA isoforms carrying distinct coding potentials","volume":"7","author":"Bal\u00e1zs","year":"2017","journal-title":"Sci. Rep."},{"key":"ref7","doi-asserted-by":"publisher","first-page":"97","DOI":"10.1016\/j.virol.2019.11.001","article-title":"Analysis of the in vitro replication phenotype of african hepatitis B virus (HBV) genotypes and subgenotypes present in Australia identifies marked differences in DNA and protein expression","volume":"540","author":"Bannister","year":"2020","journal-title":"Virology"},{"key":"ref8","doi-asserted-by":"publisher","first-page":"1022","DOI":"10.1016\/j.jhep.2013.06.018","article-title":"Hepatitis B virus splicing is enhanced prior to development of hepatocellular carcinoma","volume":"59","author":"Bayliss","year":"2013","journal-title":"J. Hepatol."},{"key":"ref9","doi-asserted-by":"publisher","first-page":"7171","DOI":"10.1128\/JVI.00243-16","article-title":"Single-molecule sequencing reveals complex genome variation of hepatitis B virus during 15 years of chronic infection following liver transplantation","volume":"90","author":"Betz-Stablein","year":"2016","journal-title":"J. Virol."},{"key":"ref10","doi-asserted-by":"publisher","first-page":"204","DOI":"10.1186\/s13059-015-0777-z","article-title":"Determining exon connectivity in complex mRNAs by nanopore sequencing","volume":"16","author":"Bolisetty","year":"2015","journal-title":"Genome Biol."},{"key":"ref11","doi-asserted-by":"publisher","first-page":"104553","DOI":"10.1016\/j.antiviral.2019.104553","article-title":"Non-invasive biomarkers for chronic hepatitis B virus infection management","volume":"169","author":"Charre","year":"2019","journal-title":"Antivir. Res."},{"key":"ref12","doi-asserted-by":"publisher","DOI":"10.1101\/2021.04.21.440736","article-title":"A systematic benchmark of nanopore long read RNA sequencing for transcript level analysis in human cell lines","author":"Chen","year":"2021","journal-title":"bioRxiv"},{"key":"ref13","doi-asserted-by":"publisher","first-page":"16459","DOI":"10.1038\/srep16459","article-title":"Hepatitis B virus spliced variants are associated with an impaired response to interferon therapy","volume":"5","author":"Chen","year":"2015","journal-title":"Sci. Rep."},{"key":"ref14","doi-asserted-by":"publisher","first-page":"156","DOI":"10.3138\/canlivj.2018-0008","article-title":"Management of hepatitis B virus infection: 2018 guidelines from the Canadian association for the study of liver disease and association of medical microbiology and infectious disease Canada","volume":"1","author":"Coffin","year":"2018","journal-title":"Can. Liver. J"},{"key":"ref15","doi-asserted-by":"publisher","first-page":"608","DOI":"10.3343\/alm.2021.41.6.608","article-title":"Concurrent nanopore next-generation sequencing of hepatitis B and delta virus genomes directly from patient plasma","volume":"41","author":"Colson","year":"2021","journal-title":"Ann. Lab. Med."},{"key":"ref16","doi-asserted-by":"publisher","first-page":"513","DOI":"10.1016\/j.cld.2006.08.009","article-title":"Do we still need a liver biopsy? Are the serum fibrosis tests ready for prime time?","volume":"10","author":"Crockett","year":"2006","journal-title":"Clin. Liver Dis."},{"key":"ref17","doi-asserted-by":"publisher","first-page":"2666","DOI":"10.1093\/bioinformatics\/bty149","article-title":"NanoPack: visualizing and processing long-read sequencing data","volume":"34","author":"De Coster","year":"2018","journal-title":"Bioinformatics"},{"key":"ref18","doi-asserted-by":"publisher","first-page":"153","DOI":"10.1186\/s13059-022-02715-w","article-title":"RATTLE: reference-free reconstruction and quantification of transcriptomes from nanopore sequencing","volume":"23","author":"de la Rubia","year":"2022","journal-title":"Genome Biol."},{"key":"ref19","doi-asserted-by":"publisher","first-page":"711733","DOI":"10.3389\/fmolb.2021.711733","article-title":"Isoform age - splice isoform profiling using long-read technologies","volume":"8","author":"De Paoli-Iseppi","year":"2021","journal-title":"Front. Mol. Biosci."},{"key":"ref20","doi-asserted-by":"publisher","first-page":"e0257521","DOI":"10.1371\/journal.pone.0257521","article-title":"Sequencing DNA with nanopores: troubles and biases","volume":"16","author":"Delahaye","year":"2021","journal-title":"PLoS One"},{"key":"ref21","doi-asserted-by":"publisher","first-page":"1520","DOI":"10.1002\/hep.32479","article-title":"Circulating HBV RNA: from biology to clinical applications","volume":"76","author":"Deng","year":"2022","journal-title":"Hepatology"},{"key":"ref22","doi-asserted-by":"publisher","first-page":"lqaa037","DOI":"10.1093\/nargab\/lqaa037","article-title":"Benchmarking of long-read correction methods","volume":"2","author":"Dohm","year":"2020","journal-title":"NAR Genom. Bioinform."},{"key":"ref23","volume-title":"SIRVsuite","author":"Drozd","year":"2022"},{"key":"ref24","doi-asserted-by":"publisher","first-page":"758613","DOI":"10.3389\/fmicb.2021.758613","article-title":"Biological characterization of hepatitis B virus genotypes: their role in viral replication and antigen expression","volume":"12","author":"Elizalde","year":"2021","journal-title":"Front. Microbiol."},{"key":"ref25","doi-asserted-by":"publisher","first-page":"5619","DOI":"10.2147\/JIR.S337561","article-title":"Alteration of the immune microenvironment in HBsAg and HBeAg dual-positive pregnant women presenting a high HBV viral load","volume":"14","author":"Gao","year":"2021","journal-title":"J. Inflamm. Res."},{"key":"ref27","doi-asserted-by":"publisher","first-page":"2095","DOI":"10.3748\/wjg.v24.i19.2095","article-title":"Detection of hyper-conserved regions in hepatitis B virus X gene potentially useful for gene therapy","volume":"24","author":"Gonz\u00e1lez","year":"2018","journal-title":"World J. Gastroenterol."},{"key":"ref28","doi-asserted-by":"publisher","first-page":"234","DOI":"10.1016\/j.jhep.2021.07.023","article-title":"A global survey of alternative splicing of HBV transcriptome using long-read sequencing","volume":"76","author":"Guan","year":"2022","journal-title":"J. Hepatol."},{"key":"ref29","doi-asserted-by":"publisher","first-page":"363","DOI":"10.1006\/viro.1997.8863","article-title":"Heterogeneity and common features of defective hepatitis B virus genomes derived from spliced pregenomic RNA","volume":"238","author":"G\u00fcnther","year":"1997","journal-title":"Virology"},{"key":"ref30","doi-asserted-by":"publisher","first-page":"792","DOI":"10.1038\/nmeth.3958","article-title":"Spliced synthetic genes as internal controls in RNA sequencing experiments","volume":"13","author":"Hardwick","year":"2016","journal-title":"Nat. Methods"},{"key":"ref31","doi-asserted-by":"publisher","first-page":"4284","DOI":"10.1128\/JVI.65.8.4284-4291.1991","article-title":"Diversity of hepatitis B virus X gene-related transcripts in hepatocellular carcinoma: a novel polyadenylation site on viral DNA","volume":"65","author":"Hilger","year":"1991","journal-title":"J. Virol."},{"key":"ref32","doi-asserted-by":"publisher","first-page":"e0076721","DOI":"10.1128\/JVI.00767-21","article-title":"MafF is an antiviral host factor that suppresses transcription from hepatitis B virus core promoter","volume":"95","author":"Ibrahim","year":"2021","journal-title":"J. Virol."},{"key":"ref33","doi-asserted-by":"publisher","first-page":"564780","DOI":"10.3389\/fgene.2021.564780","article-title":"Distinct extracellular RNA profiles in different plasma components","volume":"12","author":"Jia","year":"2021","journal-title":"Front. Genet."},{"key":"ref34","doi-asserted-by":"publisher","first-page":"14487-y","DOI":"10.1038\/s41598-021-93593-y","article-title":"Combined nanopore and single-molecule real-time sequencing survey of human betaherpesvirus 5 transcriptome","volume":"11","author":"Kakuk","year":"2021","journal-title":"Sci. Rep."},{"key":"ref35","doi-asserted-by":"publisher","first-page":"637","DOI":"10.1053\/j.gastro.2012.06.009","article-title":"Preserved T-cell function in children and young adults with immune-tolerant chronic hepatitis B","volume":"143","author":"Kennedy","year":"2012","journal-title":"Gastroenterology"},{"key":"ref36","doi-asserted-by":"publisher","first-page":"97","DOI":"10.1006\/jsre.1997.5182","article-title":"Detection of hepatitis B virus X transcripts in human hepatocellular carcinoma tissues","volume":"73","author":"Kobayashi","year":"1997","journal-title":"J. Surg. Res."},{"key":"ref37","doi-asserted-by":"publisher","first-page":"680","DOI":"10.1128\/AAC.00680-17","article-title":"Hepatitis B virus capsid assembly modulators, but not nucleoside analogs, inhibit the production of extracellular pregenomic RNA and spliced RNA variants","volume":"61","author":"Lam","year":"2017","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref26","volume-title":"SIRVs spike-in RNA variant controls","year":"2021"},{"key":"ref38","doi-asserted-by":"publisher","first-page":"mgen000492","DOI":"10.1099\/mgen.0.000492","article-title":"Quantitative analysis of the splice variants expressed by the major hepatitis B virus genotypes","volume":"7","author":"Lim","year":"2021","journal-title":"Microb. Genom."},{"key":"ref39","doi-asserted-by":"publisher","first-page":"e01275","DOI":"10.1128\/JCM.01275-19","article-title":"Diagnostic value of detection of pregenomic RNA in sera of hepatitis B virus-infected patients with different clinical outcomes","volume":"58","author":"Lin","year":"2020","journal-title":"J. Clin. Microbiol."},{"key":"ref40","doi-asserted-by":"publisher","first-page":"53","DOI":"10.1186\/s12876-019-0966-4","article-title":"Serum HBV RNA quantification: useful for monitoring natural history of chronic hepatitis B infection","volume":"19","author":"Liu","year":"2019","journal-title":"BMC Gastroenterol."},{"key":"ref41","doi-asserted-by":"publisher","first-page":"e104","DOI":"10.7717\/peerj-cs.104","article-title":"Bracken: estimating species abundance in metagenomics data","volume":"3","author":"Lu","year":"2017","journal-title":"PeerJ Comput. Sci."},{"key":"ref42","doi-asserted-by":"publisher","first-page":"604","DOI":"10.1111\/jvh.13702","article-title":"Secreted hepatitis B virus splice variants differ by HBV genotype and across phases of chronic hepatitis B infection","volume":"29","author":"Maslac","year":"2022","journal-title":"J. Viral Hepat."},{"key":"ref43","doi-asserted-by":"publisher","first-page":"39","DOI":"10.1111\/hepr.13574","article-title":"Deep sequencing analysis of serum hepatitis B virus-RNA during nucleot(s)ide analogue therapy","volume":"51","author":"Matsuda","year":"2021","journal-title":"Hepatol. Res."},{"key":"ref44","doi-asserted-by":"publisher","first-page":"7081","DOI":"10.1038\/s41598-019-43524-9","article-title":"Illumina and nanopore methods for whole genome sequencing of hepatitis B virus (HBV)","volume":"9","author":"McNaughton","year":"2019","journal-title":"Sci. Rep."},{"key":"ref45","doi-asserted-by":"publisher","first-page":"001856","DOI":"10.1099\/jgv.0.001856","article-title":"An enrichment protocol and analysis pipeline for long read sequencing of the hepatitis B virus transcriptome","volume":"104","author":"Ng","year":"2023","journal-title":"J. Gen. Virol."},{"key":"ref46","doi-asserted-by":"publisher","first-page":"606","DOI":"10.3389\/fgene.2020.00606","article-title":"Methodologies for transcript profiling using long-read technologies","volume":"11","author":"Oikonomopoulos","year":"2020","journal-title":"Front. Genet."},{"key":"ref47","doi-asserted-by":"publisher","first-page":"224","DOI":"10.1111\/jvh.13260","article-title":"Insights for clinical diagnostic indicators of virus and host in chronic hepatitis B infection","volume":"27","author":"Ou","year":"2020","journal-title":"J. Viral Hepat."},{"key":"ref48","doi-asserted-by":"publisher","first-page":"42","DOI":"10.1016\/j.jcv.2019.01.002","article-title":"Analysis of serum hepatitis B virus RNA levels in a multiethnic cohort of pregnant chronic hepatitis B carriers","volume":"111","author":"Patel","year":"2019","journal-title":"J. Clin. Virol."},{"key":"ref49","doi-asserted-by":"publisher","DOI":"10.1101\/080747","article-title":"SIRVs: spike-in RNA variants as external isoform controls in RNA-sequencing","author":"Paul","year":"2016","journal-title":"bioRxiv"},{"key":"ref50","doi-asserted-by":"publisher","first-page":"2","DOI":"10.1038\/s41467-020-20340-8","article-title":"Error correction enables use of oxford nanopore technology for reference-free transcriptome analysis","volume":"12","author":"Sahlin","year":"2021","journal-title":"Nat. Commun."},{"key":"ref51","doi-asserted-by":"publisher","first-page":"e0194366","DOI":"10.1371\/journal.pone.0194366","article-title":"Early MinION\u2122 nanopore single-molecule sequencing technology enables the characterization of hepatitis B virus genetic complexity in clinical samples","volume":"13","author":"Sauvage","year":"2018","journal-title":"PLoS One"},{"key":"ref52","doi-asserted-by":"publisher","first-page":"3","DOI":"10.1186\/1471-2199-7-3","article-title":"The RIN: an RNA integrity number for assigning integrity values to RNA measurements","volume":"7","author":"Schroeder","year":"2006","journal-title":"BMC Mol. Biol."},{"key":"ref53","doi-asserted-by":"publisher","first-page":"401","DOI":"10.1006\/viro.1996.0495","article-title":"DNA sequence requirements for the activation of a CATAAA polyadenylation signal within the hepatitis B virus X reading frame: rapid detection of truncated transcripts","volume":"223","author":"Schutz","year":"1996","journal-title":"Virology"},{"key":"ref54","doi-asserted-by":"publisher","first-page":"55","DOI":"10.1093\/dnares\/dsy038","article-title":"Evaluation and application of RNA-seq by MinION","volume":"26","author":"Seki","year":"2019","journal-title":"DNA Res."},{"key":"ref55","doi-asserted-by":"publisher","first-page":"e1008945","DOI":"10.1371\/journal.ppat.1008945","article-title":"Biogenesis and molecular characteristics of serum hepatitis B virus RNA","volume":"16","author":"Shen","year":"2020","journal-title":"PLoS Pathog."},{"key":"ref56","doi-asserted-by":"publisher","first-page":"11307","DOI":"10.1038\/ncomms11307","article-title":"Fast and sensitive mapping of nanopore sequencing reads with GraphMap","volume":"7","author":"Sovi\u0107","year":"2016","journal-title":"Nat. Commun."},{"key":"ref57","doi-asserted-by":"publisher","first-page":"40","DOI":"10.1016\/j.jhep.2020.01.028","article-title":"Full-length 5' RACE identifies all major HBV transcripts in HBV-infected hepatocytes and patient serum","volume":"73","author":"Stadelmayer","year":"2020","journal-title":"J. Hepatol."},{"key":"ref58","doi-asserted-by":"publisher","first-page":"1438","DOI":"10.1038\/s41467-020-15171-6","article-title":"Full-length transcript characterization of SF3B1 mutation in chronic lymphocytic leukemia reveals downregulation of retained introns","volume":"11","author":"Tang","year":"2020","journal-title":"Nat. Commun."},{"key":"ref59","doi-asserted-by":"publisher","first-page":"834","DOI":"10.3389\/fgene.2019.00834","article-title":"Multiple long-read sequencing survey of herpes simplex virus dynamic transcriptome","volume":"10","author":"Tomb\u00e1cz","year":"2019","journal-title":"Front. Genet."},{"key":"ref60","doi-asserted-by":"publisher","first-page":"13822","DOI":"10.1038\/s41598-020-70794-5","article-title":"Long-read assays shed new light on the transcriptome complexity of a viral pathogen","volume":"10","author":"Tomb\u00e1cz","year":"2020","journal-title":"Sci. Rep."},{"key":"ref61","doi-asserted-by":"publisher","first-page":"919","DOI":"10.3390\/pathogens10080919","article-title":"Time-course transcriptome profiling of a poxvirus using long-read full-length assay","volume":"10","author":"Tomb\u00e1cz","year":"2021","journal-title":"Pathogens"},{"key":"ref62","doi-asserted-by":"publisher","first-page":"1188","DOI":"10.1007\/s00535-012-0737-2","article-title":"Serum HBV RNA and HBeAg are useful markers for the safe discontinuation of nucleotide analogue treatments in chronic hepatitis B patients","volume":"48","author":"Tsuge","year":"2013","journal-title":"J. Gastroenterol."},{"key":"ref63","doi-asserted-by":"publisher","first-page":"69","DOI":"10.3390\/life12010069","article-title":"Direct RNA nanopore sequencing of SARS-CoV-2 extracted from critical material from swabs","volume":"12","author":"Vacca","year":"2022","journal-title":"Life"},{"key":"ref64","doi-asserted-by":"publisher","first-page":"100126","DOI":"10.1016\/j.jcvp.2022.100126","article-title":"Analytical and clinical validation of 3\u2032 RACE RT-qPCR assay for detection and quantification of hepatitis B virus (HBV) serum RNA","volume":"2","author":"Vachon","year":"2022","journal-title":"J. Clin. Virol. Plus"},{"key":"ref65","doi-asserted-by":"publisher","first-page":"66","DOI":"10.1002\/hep.27381","article-title":"Serum hepatitis B virus RNA levels as an early predictor of hepatitis B envelope antigen seroconversion during treatment with polymerase inhibitors","volume":"61","author":"van B\u00f6mmel","year":"2015","journal-title":"Hepatology"},{"key":"ref66","doi-asserted-by":"publisher","first-page":"610","DOI":"10.1111\/jvh.13272","article-title":"Serum hepatitis B virus RNA predicts response to peginterferon treatment in HBeAg-positive chronic hepatitis B","volume":"27","author":"van Campenhout","year":"2020","journal-title":"J. Viral Hepat."},{"key":"ref67","doi-asserted-by":"publisher","first-page":"700","DOI":"10.1016\/j.jhep.2016.05.029","article-title":"Serum hepatitis B virus RNA is encapsidated pregenome RNA that may be associated with persistence of viral infection and rebound","volume":"65","author":"Wang","year":"2016","journal-title":"J. Hepatol."},{"key":"ref68","volume-title":"Filtlong","author":"Wick","year":"2022"},{"key":"ref69","doi-asserted-by":"publisher","first-page":"883","DOI":"10.1111\/apt.12658","article-title":"Coincidental metabolic syndrome increases the risk of liver fibrosis progression in patients with chronic hepatitis B--a prospective cohort study with paired transient elastography examinations","volume":"39","author":"Wong","year":"2014","journal-title":"Aliment. Pharmacol. Ther."},{"key":"ref70","doi-asserted-by":"publisher","first-page":"257","DOI":"10.1186\/s13059-019-1891-0","article-title":"Improved metagenomic analysis with kraken 2","volume":"20","author":"Wood","year":"2019","journal-title":"Genome Biol."},{"key":"ref71","doi-asserted-by":"publisher","first-page":"1297","DOI":"10.1038\/s41592-019-0617-2","article-title":"Nanopore native RNA sequencing of a human poly(a) transcriptome","volume":"16","author":"Workman","year":"2019","journal-title":"Nat. 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