Patients with colorectal cancer (CRC) have a different gut microbiome signature than individuals without CRC. Little is known about the viral component of CRC-associated microbiome. We aimed to identify and validate viral taxonomic markers of CRC that might be used in detection of the disease or predicting outcome. We performed shotgun metagenomic analyses of viromes of fecal samples from 74 patients with CRC (cases) and 92 individuals without CRC (controls) in Hong Kong (discovery cohort). Viral sequences were classified by taxonomic alignment against an integrated microbial reference genome database. Viral markers associated with CRC were validated using fecal samples from 3 separate cohorts: 111 patients with CRC and 112 controls in Hong Kong, 46 patients with CRC and 63 controls in Austria, and 91 patients with CRC and 66 controls in France and Germany. Using abundance profiles of CRC-associated virome genera, we constructed random survival forest models to identify those associated with patient survival times. The diversity of the gut bacteriophage community was significantly increased in patients with CRC compared with controls. Twenty-two viral taxa discriminated cases from controls with an area under the receiver operating characteristic curve of 0.802 in the discovery cohort. The viral markers were validated in 3 cohorts, with area under the receiver operating characteristic curves of 0.763, 0.736, and 0.715, respectively. Clinical subgroup analysis showed that dysbiosis of the gut virome was associated with early- and late-stage CRC. A combination of 4 taxonomic markers associated with reduced survival of patients with CRC (log-rank test, P = 8.1 × 10^–6) independently of tumor stage, lymph node metastases, or clinical parameters. We found altered interactions between bacteriophages and oral bacterial commensals in fecal samples from patients with CRC compared with controls. In a metagenomic analysis of fecal samples from patients and controls, we identified virome signatures associated with CRC. These data might be used to develop tools to identify individuals with CRC or predict outcomes.

The outbreak of COVID-19 has positively impacted the NGS market recently. Targeted sequencing (TS) has become an important routine technique in both clinical and research settings, with advantages including high confidence and accuracy, a reasonable turnaround time, relatively low cost, and fewer data burdens with the level of bioinformatics or computational demand. Since there are no clear consensus guidelines on the wide range of next-generation sequencing (NGS) platforms and techniques, there is a vital need for researchers and clinicians to develop efficient approaches, especially for the molecular diagnosis of diseases in the emergency of the disease and the global pandemic outbreak of COVID-19. In this review, we aim to summarize different methods of TS, demonstrate parameters for TS assay designs, illustrate different TS panels, discuss their limitations, and present the challenges of TS concerning their clinical application for the molecular diagnosis of human diseases.

Alternative splicing (AS) allows generation of cell type–specific mRNA transcripts and contributes to hallmarks of cancer. Genome‐wide analysis for AS in human hepatocellular carcinoma (HCC), however, is limited. We sought to obtain a comprehensive AS landscape in HCC and define tumor‐associated variants. Single‐molecule real‐time long‐read RNA sequencing was performed on patient‐derived HCC cells, and presence of splice junctions was defined by SpliceMap‐LSC‐IDP algorithm. We obtained an all‐inclusive map of annotated AS variants and further discovered 362 alternative spliced variants that are not previously reported in any database (neither RefSeq nor GENCODE). They were mostly derived from intron retention and early termination codon with an in‐frame open reading frame in 81.5%. We corroborated many of these predicted unannotated and annotated variants to be tumor specific in an independent cohort of primary HCC tumors and matching nontumoral liver. Using the combined Sanger sequencing and TaqMan junction assays, unique and common expressions of spliced variants including enzyme regulators (ARHGEF2, SERPINH1), chromatin modifiers (DEK, CDK9, RBBP7), RNA‐binding proteins (SRSF3, RBM27, MATR3, YBX1), and receptors (ADRM1, CD44v8‐10, vitamin D receptor, ROR1) were determined in HCC tumors. We further focused functional investigations on ARHGEF2 variants (v1 and v3) that arise from the common amplified site chr.1q22 of HCC. Their biological significance underscores two major cancer hallmarks, namely cancer stemness and epithelial‐to‐mesenchymal transition–mediated cell invasion and migration, although v3 is consistently more potent than v1. Conclusion: Alternative isoforms and tumor‐specific isoforms that arise from aberrant splicing are common during the liver tumorigenesis. Our results highlight insights gained from the analysis of AS in HCC.

Summary: Insertional mutagenesis from virus infection is an important pathogenic risk for the development of cancer. Despite the advent of high-throughput sequencing, discovery of viral integration sites and expressed viral fusion events are still limited. Here, we present ViralFusionSeq (VFS), which combines soft-clipping information, read-pair analysis and targeted de novo assembly to discover and annotate viral–human fusions. VFS was used in an RNA-Seq experiment, simulated DNA-Seq experiment and re-analysis of published DNA-Seq datasets. Our experiments demonstrated that VFS is both sensitive and highly accurate. Availability: VFS is distributed under GPL version 3 at http://hkbic.cuhk.edu.hk/software/viralfusionseq Contact: tf.chan@cuhk.edu.hk Supplementary information: Supplementary data are available at Bioinformatics Online

Background: Spinocerebellar ataxias (SCAs) are a group of clinically and genetically diverse and autosomal-dominant disorders characterised by neurological deficits in the cerebellum. At present, there is no cure for SCAs. Of the different distinct subtypes of autosomal-dominant SCAs identified to date, causative genes for only a fraction of them are currently known. In this study, we investigated the cause of an autosomal-dominant SCA phenotype in a family that exhibits cerebellar ataxia and pontocerebellar atrophy along with a global reduction in brain volume. Methods and results: Whole-exome analysis revealed a missense mutation c.G1391A (p.R464H) in the coding region of the coiled-coil domain containing 88C (CCDC88C) gene in all affected individuals. Functional studies showed that the mutant form of CCDC88C activates the c-Jun N-terminal kinase (JNK) pathway, induces caspase 3 cleavage and triggers apoptosis. Conclusions: This study expands our understanding of the cause of autosomal-dominant SCAs, a group of heterogeneous congenital neurological conditions in humans, and unveils a link between the JNK stress pathway and cerebellar atrophy.