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Analysis of mutational signatures is becoming routine in cancer genomics, with implications for pathogenesis, classification, prognosis, and even treatment decisions. However, the field lacks a consensus on analysis and result interpretation. Using whole-genome sequencing of multiple myeloma (MM), chronic lymphocytic leukemia (CLL) and acute myeloid leukemia, we compare the performance of public signature analysis tools. We describe caveats and pitfalls of de novo signature extraction and fitting approaches, reporting on common inaccuracies: erroneous signature assignment, identification of localized hyper-mutational processes, overcalling of signatures. We provide reproducible solutions to solve these issues and use orthogonal approaches to validate our results. We show how a comprehensive mutational signature analysis may provide relevant biological insights, reporting evidence of c-AID activity among unmutated CLL cases or the absence of BRCA1/BRCA2-mediated homologous recombination deficiency in a MM cohort. Finally, we propose a general analysis framework to ensure production of accurate and reproducible mutational signature data. Mutational signature analysis provides important information about the mutational processes underpinning different stages of tumorigenesis. Here, the authors compare publicly available signature extraction tools and suggest a framework for the generation of accurate and reproducible signature data.

Immunoglobulin (Ig) gene rearrangements and oncogenic translocations are routinely assessed during the characterization of B cell neoplasms and stratification of patients with distinct clinical and biological features, with the assessment done using Sanger sequencing, targeted next-generation sequencing, or fluorescence in situ hybridization (FISH). Currently, a complete Ig characterization cannot be extracted from whole-genome sequencing (WGS) data due to the inherent complexity of the Ig loci. Here, we introduce IgCaller, an algorithm designed to fully characterize Ig gene rearrangements and oncogenic translocations from short-read WGS data. Using a cohort of 404 patients comprising different subtypes of B cell neoplasms, we demonstrate that IgCaller identifies both heavy and light chain rearrangements to provide additional information on their functionality, somatic mutational status, class switch recombination, and oncogenic Ig translocations. Our data thus support IgCaller to be a reliable alternative to Sanger sequencing and FISH for studying the genetic properties of the Ig loci. Immunoglobulin (Ig) rearrangement and translocation information are usually obtained by targeted sequencing of the respective loci. Here, the authors present the IgCaller algorithm, which extracts Ig heavy and light chain genetic properties from short-read whole-genome sequencing results to provide a feasible alternative to direct sequencing.

Background One of the hallmarks of cancer is the disruption of gene expression patterns. Many molecular lesions contribute to this phenotype, and the importance of aberrant DNA methylation profiles is increasingly recognized. Much of the research effort in this area has examined proximal promoter regions and epigenetic alterations at other loci are not well characterized. Results Using whole genome bisulfite sequencing to examine uncharted regions of the epigenome, we identify a type of far-reaching DNA methylation alteration in cancer cells of the distal regulatory sequences described as super-enhancers. Human tumors undergo a shift in super-enhancer DNA methylation profiles that is associated with the transcriptional silencing or the overactivation of the corresponding target genes. Intriguingly, we observe locally active fractions of super-enhancers detectable through hypomethylated regions that suggest spatial variability within the large enhancer clusters. Functionally, the DNA methylomes obtained suggest that transcription factors contribute to this local activity of super-enhancers and that trans -acting factors modulate DNA methylation profiles with impact on transforming processes during carcinogenesis. Conclusions We develop an extensive catalogue of human DNA methylomes at base resolution to better understand the regulatory functions of DNA methylation beyond those of proximal promoter gene regions. CpG methylation status in normal cells points to locally active regulatory sites at super-enhancers, which are targeted by specific aberrant DNA methylation events in cancer, with putative effects on the expression of downstream genes.

A new approach overcomes the hurdles of identifying large, complex structural variants in cancer genomes by directly comparing tumor and normal genome sequencing reads. The development of high-throughput sequencing technologies has advanced our understanding of cancer. However, characterizing somatic structural variants in tumor genomes is still challenging because current strategies depend on the initial alignment of reads to a reference genome. Here, we describe SMUFIN (somatic mutation finder), a single program that directly compares sequence reads from normal and tumor genomes to accurately identify and characterize a range of somatic sequence variation, from single-nucleotide variants (SNV) to large structural variants at base pair resolution. Performance tests on modeled tumor genomes showed average sensitivity of 92% and 74% for SNVs and structural variants, with specificities of 95% and 91%, respectively. Analyses of aggressive forms of solid and hematological tumors revealed that SMUFIN identifies breakpoints associated with chromothripsis and chromoplexy with high specificity. SMUFIN provides an integrated solution for the accurate, fast and comprehensive characterization of somatic sequence variation in cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

José Martin-Subero and colleagues report whole-genome bisulfite sequencing and methylome analysis of two CLLs and three B-cell subpopulations using high-density microarrays on 139 CLLs. They identify widespread hypomethylation in the gene body that is largely associated with intragenic enhancer elements. We have extensively characterized the DNA methylomes of 139 patients with chronic lymphocytic leukemia (CLL) with mutated or unmutated IGHV and of several mature B-cell subpopulations through the use of whole-genome bisulfite sequencing and high-density microarrays. The two molecular subtypes of CLL have differing DNA methylomes that seem to represent epigenetic imprints from distinct normal B-cell subpopulations. DNA hypomethylation in the gene body, targeting mostly enhancer sites, was the most frequent difference between naive and memory B cells and between the two molecular subtypes of CLL and normal B cells. Although DNA methylation and gene expression were poorly correlated, we identified gene-body CpG dinucleotides whose methylation was positively or negatively associated with expression. We have also recognized a DNA methylation signature that distinguishes new clinico-biological subtypes of CLL. We propose an epigenomic scenario in which differential methylation in the gene body may have functional and clinical implications in leukemogenesis.

Chronic lymphocytic leukemia (CLL) is a frequent hematological neoplasm in which underlying epigenetic alterations are only partially understood. Here, we analyze the reference epigenome of seven primary CLLs and the regulatory chromatin landscape of 107 primary cases in the context of normal B cell differentiation. We identify that the CLL chromatin landscape is largely influenced by distinct dynamics during normal B cell maturation. Beyond this, we define extensive catalogues of regulatory elements de novo reprogrammed in CLL as a whole and in its major clinico-biological subtypes classified by IGHV somatic hypermutation levels. We uncover that IGHV-unmutated CLLs harbor more active and open chromatin than IGHV-mutated cases. Furthermore, we show that de novo active regions in CLL are enriched for NFAT, FOX and TCF/LEF transcription factor family binding sites. Although most genetic alterations are not associated with consistent epigenetic profiles, CLLs with MYD88 mutations and trisomy 12 show distinct chromatin configurations. Furthermore, we observe that non-coding mutations in IGHV-mutated CLLs are enriched in H3K27ac-associated regulatory elements outside accessible chromatin. Overall, this study provides an integrative portrait of the CLL epigenome, identifies extensive networks of altered regulatory elements and sheds light on the relationship between the genetic and epigenetic architecture of the disease. An integrated resource of (epi)genomic features in annotated chronic lymphocytic leukemia (CLL) primary samples uncovers subgroup-specific regulatory alterations associated with clinical behavior.