CSI NGS Portal: An Online Platform for Automated NGS Data Analysis and Sharing.

Next-generation sequencing (NGS) has been a widely-used technology in biomedical research for understanding the role of molecular genetics of cells in health and disease. A variety of computational tools have been developed to analyse the vastly growing NGS data, which often require bioinformatics skills, tedious work and a significant amount of time. To facilitate data processing steps minding the gap between biologists and bioinformaticians, we developed CSI NGS Portal, an online platform which gathers established bioinformatics pipelines to provide fully automated NGS data analysis and sharing in a user-friendly website. The portal currently provides 16 standard pipelines for analysing data from DNA, RNA, smallRNA, ChIP, RIP, 4C, SHAPE, circRNA, eCLIP, Bisulfite and scRNA sequencing, and is flexible to expand with new pipelines. The users can upload raw data in FASTQ format and submit jobs in a few clicks, and the results will be self-accessible via the portal to view/download/share in real-time. The output can be readily used as the final report or as input for other tools depending on the pipeline. Overall, CSI NGS Portal helps researchers rapidly analyse their NGS data and share results with colleagues without the aid of a bioinformatician. The portal is freely available at: https://csibioinfo.nus.edu.sg/csingsportal.

The c-MYC-BMI1 axis is essential for SETDB1-mediated breast tumourigenesis.

Characterising the activated oncogenic signalling that leads to advanced breast cancer is of clinical importance. Here, we showed that SET domain, bifurcated 1 (SETDB1), a histone H3 lysine 9 methyltransferase, is aberrantly expressed and behaves as an oncogenic driver in breast cancer. SETDB1 enhances c-MYC and cyclin D1 expression by promoting the internal ribosome entry site (IRES)-mediated translation of MYC/CCND1 mRNA, resulting in prominent signalling of c-MYC to promote cell cycle progression, and provides a growth/self-renewal advantage to breast cancer cells. The activated c-MYC-BMI1 axis is essential for SETDB1-mediated breast tumourigenesis, because silencing of either c-MYC or BMI1 profoundly impairs the enhanced growth/colony formation conferred by SETDB1. Furthermore, c-MYC directly binds to the SETDB1 promoter region and enhances its transcription, suggesting a positive regulatory interplay between SETDB1 and c-MYC. In this study, we identified SETDB1 as a prominent oncogene and characterised the underlying mechanism whereby SETDB1 drives breast cancer, providing a therapeutic rationale for targeting SETDB1-BMI1 signalling in breast cancer. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

An RNA editing/dsRNA binding-independent gene regulatory mechanism of ADARs and its clinical implication in cancer.

Adenosine-to-inosine (A-to-I) RNA editing, catalyzed by Adenosine DeAminases acting on double-stranded RNA(dsRNA) (ADAR), occurs predominantly in the 3' untranslated regions (3'UTRs) of spliced mRNA. Here we uncover an unanticipated link between ADARs (ADAR1 and ADAR2) and the expression of target genes undergoing extensive 3'UTR editing. Using METTL7A (Methyltransferase Like 7A), a novel tumor suppressor gene with multiple editing sites at its 3'UTR, we demonstrate that its expression could be repressed by ADARs beyond their RNA editing and double-stranded RNA (dsRNA) binding functions. ADARs interact with Dicer to augment the processing of pre-miR-27a to mature miR-27a. Consequently, mature miR-27a targets the METTL7A 3'UTR to repress its expression level. In sum, our study unveils that the extensive 3'UTR editing of METTL7A is merely a footprint of ADAR binding, and there are a subset of target genes that are equivalently regulated by ADAR1 and ADAR2 through their non-canonical RNA editing and dsRNA binding-independent functions, albeit maybe less common. The functional significance of ADARs is much more diverse than previously appreciated and this gene regulatory function of ADARs is most likely to be of high biological importance beyond the best-studied editing function. This non-editing side of ADARs opens another door to target cancer.

Profiling the B/T cell receptor repertoire of lymphocyte derived cell lines.

BACKGROUND: Clonal VDJ rearrangement of B/T cell receptors (B/TCRs) occurring during B/T lymphocyte development has been used as a marker to track the clonality of B/T cell populations. METHODS: We systematically profiled the B/T cell receptor repertoire of 936 cancer cell lines across a variety of cancer types as well as 462 Epstein-Barr Virus (EBV) transformed normal B lymphocyte lines using RNA sequencing data. RESULTS: Rearranged B/TCRs were readily detected in cell lines derived from lymphocytes, and subclonality or potential biclonality were found in a number of blood cancer cell lines. Clonal BCR/TCR rearrangements were detected in several blast phase CML lines and unexpectedly, one gastric cancer cell line (KE-97), reflecting a lymphoid origin of these cells. Notably, clonality was highly prevalent in EBV transformed B lymphocytes, suggesting either transformation only occurred in a few B cells or those with a growth advantage dominated the transformed population through clonal evolution. CONCLUSIONS: Our analysis reveals the complexity and heterogeneity of the BCR/TCR rearrangement repertoire and provides a unique insight into the clonality of lymphocyte derived cell lines.

ADAR-Mediated RNA Editing Predicts Progression and Prognosis of Gastric Cancer.

BACKGROUD & AIMS: Gastric cancer (GC) is the third leading cause of global cancer mortality. Adenosine-to-inosine RNA editing is a recently described novel epigenetic mechanism involving sequence alterations at the RNA but not DNA level, primarily mediated by ADAR (adenosine deaminase that act on RNA) enzymes. Emerging evidence suggests a role for RNA editing and ADARs in cancer, however, the relationship between RNA editing and GC development and progression remains unknown. METHODS: In this study, we leveraged on the next-generation sequencing transcriptomics to demarcate the GC RNA editing landscape and the role of ADARs in this deadly malignancy. RESULTS: Relative to normal gastric tissues, almost all GCs displayed a clear RNA misediting phenotype with ADAR1/2 dysregulation arising from the genomic gain and loss of the ADAR1 and ADAR2 gene in primary GCs, respectively. Clinically, patients with GCs exhibiting ADAR1/2 imbalance demonstrated extremely poor prognoses in multiple independent cohorts. Functionally, we demonstrate in vitro and in vivo that ADAR-mediated RNA misediting is closely associated with GC pathogenesis, with ADAR1 and ADAR2 playing reciprocal oncogenic and tumor suppressive roles through their catalytic deaminase domains, respectively. Using an exemplary target gene PODXL (podocalyxin-like), we demonstrate that the ADAR2-regulated recoding editing at codon 241 (His to Arg) confers a loss-of-function phenotype that neutralizes the tumorigenic ability of the unedited PODXL. CONCLUSIONS: Our study highlights a major role for RNA editing in GC disease and progression, an observation potentially missed by previous next-generation sequencing analyses of GC focused on DNA alterations alone. Our findings also suggest new GC therapeutic opportunities through ADAR1 enzymatic inhibition or the potential restoration of ADAR2 activity.

Profiling of somatic mutations in acute myeloid leukemia with FLT3-ITD at diagnosis and relapse.

Acute myeloid leukemia (AML) with an FLT3 internal tandem duplication (FLT3-ITD) mutation is an aggressive hematologic malignancy with a grave prognosis. To identify the mutational spectrum associated with relapse, whole-exome sequencing was performed on 13 matched diagnosis, relapse, and remission trios followed by targeted sequencing of 299 genes in 67 FLT3-ITD patients. The FLT3-ITD genome has an average of 13 mutations per sample, similar to other AML subtypes, which is a low mutation rate compared with that in solid tumors. Recurrent mutations occur in genes related to DNA methylation, chromatin, histone methylation, myeloid transcription factors, signaling, adhesion, cohesin complex, and the spliceosome. Their pattern of mutual exclusivity and cooperation among mutated genes suggests that these genes have a strong biological relationship. In addition, we identified mutations in previously unappreciated genes such as MLL3, NSD1, FAT1, FAT4, and IDH3B. Mutations in 9 genes were observed in the relapse-specific phase. DNMT3A mutations are the most stable mutations, and this DNMT3A-transformed clone can be present even in morphologic complete remissions. Of note, all AML matched trio samples shared at least 1 genomic alteration at diagnosis and relapse, suggesting common ancestral clones. Two types of clonal evolution occur at relapse: either the founder clone recurs or a subclone of the founder clone escapes from induction chemotherapy and expands at relapse by acquiring new mutations. Relapse-specific mutations displayed an increase in transversions. Functional assays demonstrated that both MLL3 and FAT1 exert tumor-suppressor activity in the FLT3-ITD subtype. An inhibitor of XPO1 synergized with standard AML induction chemotherapy to inhibit FLT3-ITD growth. This study clearly shows that FLT3-ITD AML requires additional driver genetic alterations in addition to FLT3-ITD alone.

Efficient production of superior dumbbell-shaped DNA minimal vectors for small hairpin RNA expression.

Genetic therapy holds great promise for the treatment of inherited or acquired genetic diseases; however, its breakthrough is hampered by the lack of suitable gene delivery systems. Dumbbell-shaped DNA minimal vectors represent an attractive, safe alternative to the commonly used viral vectors which are fraught with risk, but dumbbell generation appears to be costly and time-consuming. We developed a new PCR-based method for dumbbell production which comprises only two steps. First, PCR amplification of the therapeutic expression cassette using chemically modified primers to form a ready-to-ligate DNA structure; and second, a highly efficient intramolecular ligation reaction. Compared with conventional strategies, the new method produces dumbbell vectors more rapidly, with higher yields and purity, and at lower costs. In addition, such produced small hairpin RNA expressing dumbbells triggered superior target gene knockdown compared with conventionally produced dumbbells or plasmids. Our novel method is suitable for large-scale dumbbell production and can facilitate clinical applications of this vector system.

Neotelomeres and telomere-spanning chromosomal arm fusions in cancer genomes revealed by long-read sequencing.

Alterations in the structure and location of telomeres are pivotal in cancer genome evolution. Here, we applied both long-read and short-read genome sequencing to assess telomere repeat-containing structures in cancers and cancer cell lines. Using long-read genome sequences that span telomeric repeats, we defined four types of telomere repeat variations in cancer cells: neotelomeres where telomere addition heals chromosome breaks, chromosomal arm fusions spanning telomere repeats, fusions of neotelomeres, and peri-centromeric fusions with adjoined telomere and centromere repeats. These results provide a framework for the systematic study of telomeric repeats in cancer genomes, which could serve as a model for understanding the somatic evolution of other repetitive genomic elements.

Human telomere length is chromosome end-specific and conserved across individuals.

Short telomeres cause age-related disease, and long telomeres contribute to cancer; however, the mechanisms regulating telomere length are unclear. We developed a nanopore-based method, which we call Telomere Profiling, to determine telomere length at nearly single-nucleotide resolution. Mapping telomere reads to chromosome ends showed chromosome end-specific length distributions that could differ by more than six kilobases. Examination of telomere lengths in 147 individuals revealed that certain chromosome ends were consistently longer or shorter. The same rank order was found in newborn cord blood, suggesting that telomere length is determined at birth and that chromosome end-specific telomere length differences are maintained as telomeres shorten with age. Telomere Profiling makes precision investigation of telomere length widely accessible for laboratory, clinical, and drug discovery efforts and will allow deeper insights into telomere biology.

Human telomere length is chromosome specific and conserved across individuals.

Short telomeres cause age-related disease and long telomeres predispose to cancer; however, the mechanisms regulating telomere length are unclear. To probe these mechanisms, we developed a nanopore sequencing method, Telomere Profiling, that is easy to implement, precise, and cost effective with broad applications in research and the clinic. We sequenced telomeres from individuals with short telomere syndromes and found similar telomere lengths to the clinical FlowFISH assay. We mapped telomere reads to specific chromosome end and identified both chromosome end-specific and haplotype-specific telomere length distributions. In the T2T HG002 genome, where the average telomere length is 5kb, we found a remarkable 6kb difference in lengths between some telomeres. Further, we found that specific chromosome ends were consistently shorter or longer than the average length across 147 individuals. The presence of conserved chromosome end-specific telomere lengths suggests there are new paradigms in telomere biology that are yet to be explored. Understanding the mechanisms regulating length will allow deeper insights into telomere biology that can lead to new approaches to disease.

The genome sequence of the Large Scabious Mining Bee, Andrena hattorfiana (Fabricius, 1775).

We present a genome assembly from an individual female Andrena hattorfiana (the Large Scabious Mining Bee; Arthropoda; Insecta; Hymenoptera; Andrenidae). The genome sequence is 428.5 megabases in span. Most of the assembly is scaffolded into seven chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 22.7 kilobases in length. Gene annotation of this assembly on Ensembl identified 11,349 protein coding genes.

Neotelomeres and Telomere-Spanning Chromosomal Arm Fusions in Cancer Genomes Revealed by Long-Read Sequencing.

Alterations in the structure and location of telomeres are key events in cancer genome evolution. However, previous genomic approaches, unable to span long telomeric repeat arrays, could not characterize the nature of these alterations. Here, we applied both long-read and short-read genome sequencing to assess telomere repeat-containing structures in cancers and cancer cell lines. Using long-read genome sequences that span telomeric repeat arrays, we defined four types of telomere repeat variations in cancer cells: neotelomeres where telomere addition heals chromosome breaks, chromosomal arm fusions spanning telomere repeats, fusions of neotelomeres, and peri-centromeric fusions with adjoined telomere and centromere repeats. Analysis of lung adenocarcinoma genome sequences identified somatic neotelomere and telomere-spanning fusion alterations. These results provide a framework for systematic study of telomeric repeat arrays in cancer genomes, that could serve as a model for understanding the somatic evolution of other repetitive genomic elements.

Identifying and correcting repeat-calling errors in nanopore sequencing of telomeres.

Nanopore long-read sequencing is an emerging approach for studying genomes, including long repetitive elements like telomeres. Here, we report extensive basecalling induced errors at telomere repeats across nanopore datasets, sequencing platforms, basecallers, and basecalling models. We find that telomeres in many organisms are frequently miscalled. We demonstrate that tuning of nanopore basecalling models leads to improved recovery and analysis of telomeric regions, with minimal negative impact on other genomic regions. We highlight the importance of verifying nanopore basecalls in long, repetitive, and poorly defined regions, and showcase how artefacts can be resolved by improvements in nanopore basecalling models.

Repurposing RNA sequencing for discovery of RNA modifications in clinical cohorts.

The study of RNA modifications in large clinical cohorts can reveal relationships between the epitranscriptome and human diseases, although this is especially challenging. We developed ModTect (https://github.com/ktan8/ModTect), a statistical framework to identify RNA modifications de novo by standard RNA-sequencing with deletion and mis-incorporation signals. We show that ModTect can identify both known (N 1-methyladenosine) and previously unknown types of mRNA modifications (N 2,N 2-dimethylguanosine) at nucleotide-resolution. Applying ModTect to 11,371 patient samples and 934 cell lines across 33 cancer types, we show that the epitranscriptome was dysregulated in patients across multiple cancer types and was additionally associated with cancer progression and survival outcomes. Some types of RNA modification were also more disrupted than others in patients with cancer. Moreover, RNA modifications contribute to multiple types of RNA-DNA sequence differences, which unexpectedly escape detection by Sanger sequencing. ModTect can thus be used to discover associations between RNA modifications and clinical outcomes in patient cohorts.

Haplotype-resolved germline and somatic alterations in renal medullary carcinomas.

BACKGROUND: Renal medullary carcinomas (RMCs) are rare kidney cancers that occur in adolescents and young adults of African ancestry. Although RMC is associated with the sickle cell trait and somatic loss of the tumor suppressor, SMARCB1, the ancestral origins of RMC remain unknown. Further, characterization of structural variants (SVs) involving SMARCB1 in RMC remains limited. METHODS: We used linked-read genome sequencing to reconstruct germline and somatic haplotypes in 15 unrelated patients with RMC registered on the Children's Oncology Group (COG) AREN03B2 study between 2006 and 2017 or from our prior study. We performed fine-mapping of the HBB locus and assessed the germline for cancer predisposition genes. Subsequently, we assessed the tumor samples for mutations outside of SMARCB1 and integrated RNA sequencing to interrogate the structural variants at the SMARCB1 locus. RESULTS: We find that the haplotype of the sickle cell mutation in patients with RMC originated from three geographical regions in Africa. In addition, fine-mapping of the HBB locus identified the sickle cell mutation as the sole candidate variant. We further identify that the SMARCB1 structural variants are characterized by blunt or 1-bp homology events. CONCLUSIONS: Our findings suggest that RMC does not arise from a single founder population and that the HbS allele is a strong candidate germline allele which confers risk for RMC. Furthermore, we find that the SVs that disrupt SMARCB1 function are likely repaired by non-homologous end-joining. These findings highlight how haplotype-based analyses using linked-read genome sequencing can be applied to identify potential risk variants in small and rare disease cohorts and provide nucleotide resolution to structural variants.

Profiling of gastric cancer cell-surface markers to achieve tumour-normal discrimination.

BACKGROUND: Differentiating between malignant and normal cells within tissue samples is vital for molecular profiling of cancer using advances in genomics and transcriptomics. Cell-surface markers of tumour-normal discrimination have additional value in terms of translatability to diagnostic and therapeutic strategies. In gastric cancer (GC), previous studies have identified individual genes or proteins that are upregulated in cancer. However, a systematic analysis of cell-surface markers and development of a composite panel involving multiple candidates to differentiate tumour from normal has not been previously reported. METHODS: Whole transcriptome sequencing (WTS) of GC and matched normal samples from the Singapore Gastric Cancer Consortium (SGCC) was used as a discovery cohort to identify upregulated putative cell-surface proteins. Matched WTS data from the The Cancer Genome Atlas (TCGA) was used as a validation cohort. Promising candidates from this analysis were validated orthogonally using multispectral immunohistochemistry (mIHC) with automated quantitative analysis using the Vectra platform. mIHC was performed on a tissue microarray containing matched normal, marginal and tumour tissues. The receiver-operating characteristic (ROC) curves were analysed to identify markers with the highest diagnostic validity independently and in combination. RESULTS: Analysis of putative membrane protein transcripts from the SGCC discovery cohort WTS data (n=15 matched tumour and normal pairs) identified several differentially and highly expressed candidates in tumour compared with normal tissues. After validation with TCGA data (n=29 matched tumour and normal pairs), the following proteins were selected for mIHC analysis: CEACAM5, CEACAM6, CLDN4, CLDN7, and EpCAM. These were compared with established glycoprotein markers in GC, namely CA19-9 and CA72-4. Individual ROC curves yielded the best performance for CEACAM5 (area under the ROC curve (AUC)=0.80), CEACAM6 (AUC=0.82), EpCAM (AUC=0.83), and CA72-4 (AUC=0.76). Combined multiplexed imaging of these four markers revealed improved specificity and sensitivity for detection of tumour from normal tissue (AUC of 4-plex=0.91). CONCLUSION: CEAMCAM5, CEACAM6, EpCAM, and CA72-4 form a versatile set of markers for robust discrimination of GC from adjacent normal tissue. As cell-surface markers, they are compatible with both IHC and live imaging approaches. These candidates may be exploited to improve automated identification of tumour tissue in GC.

Next Generation Sequencing Reveals Novel Mutations in Mismatch Repair Genes and Other Cancer Predisposition Genes in Asian Patients with Suspected Lynch Syndrome.

BACKGROUND: Although at least 5 genes are implicated in Lynch Syndrome (LS), up to 50% of suspected cases are owing to undefined genes. We utilized next generation sequencing (NGS) to characterize the mutation profile of patients with cancer (CA) suspected to have LS. PATIENTS AND METHODS: We enrolled 174 Asian patients with CA from our CA Genetics Clinic from 2000 to 2014 suspected to have LS, and obtained germline DNA for NGS using TruSight Cancer. Frameshift, nonsense, and known deleterious mutations were considered pathogenic. Polymorphisms ≤ 1% frequency in 1000 Genomes (Asian) were classified using established databases. RESULTS: Of the 174 probands, 80.5% were Chinese, the median age at CA diagnosis was 45 years (range, 18-82 years), and 84.5% and 8.6% had colon and LS-like CA, respectively. Forty-seven of 100 evaluable colon CA probands had LS-like histopathologic features. Nineteen of 174 had family history fulfilling Amsterdam I/II Criteria, whereas the rest fulfilled Bethesda Guidelines. Thirty-one of 174 harbored pathogenic mutations with 10 in LS genes only, 20 in non-LS genes only, and 1 in both. Of the 11 with LS gene mutations, MLH1 was most commonly involved (n = 7), followed by MSH2, MSH6, and PMS2. Nine of 174 had pathogenic mutations diagnostic of alternative hereditary syndromes including 2 each in CDH1, APC, and BRCA1, and 1 each in BRCA2, SMAD4, and MUTYH. Ten unique mutations were detected in low-to-moderate penetrance genes: 6 individuals had a recurring novel KIT:c.2836C>T nonsense mutation (n = 3) or ERCC4:c.2169C>A nonsense mutation (n = 3) without LS gene mutation, which is of clinical interest. CONCLUSIONS: In this Asian study, NGS proved to be feasible in screening for causative mutations in patients with CA suspected to have LS.

Lineage-specific RUNX2 super-enhancer activates MYC and promotes the development of blastic plasmacytoid dendritic cell neoplasm.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive subtype of acute leukemia, the cell of origin of which is considered to be precursors of plasmacytoid dendritic cells (pDCs). Since translocation (6;8)(p21;q24) is a recurrent anomaly for BPDCN, we demonstrate that a pDC-specific super-enhancer of RUNX2 is associated with the MYC promoter due to t(6;8). RUNX2 ensures the expression of pDC-signature genes in leukemic cells, but also confers survival and proliferative properties in BPDCN cells. Furthermore, the pDC-specific RUNX2 super-enhancer is hijacked to activate MYC in addition to RUNX2 expression, thereby promoting the proliferation of BPDCN. We also demonstrate that the transduction of MYC and RUNX2 is sufficient to initiate the transformation of BPDCN in mice lacking Tet2 and Tp53, providing a model that accurately recapitulates the aggressive human disease and gives an insight into the molecular mechanisms underlying the pathogenesis of BPDCN.