Widespread Aberrant Alternative Splicing despite Molecular Remission in Chronic Myeloid Leukaemia Patients

Vast transcriptomics and epigenomics changes are characteristic of human cancers, including leukaemia. At remission, we assume that these changes normalise so that omics-profiles resemble those of healthy individuals. However, an in-depth transcriptomic and epigenomic analysis of cancer remission has not been undertaken. A striking exemplar of targeted remission induction occurs in chronic myeloid leukaemia (CML) following tyrosine kinase inhibitor (TKI) therapy. Using RNA sequencing and whole-genome bisulfite sequencing, we profiled samples from chronic-phase CML patients at diagnosis and remission and compared these to healthy donors. Remarkably, our analyses revealed that abnormal splicing distinguishes remission samples from normal controls. This phenomenon is independent of the TKI drug used and in striking contrast to the normalisation of gene expression and DNA methylation patterns. Most remarkable are the high intron retention (IR) levels that even exceed those observed in the diagnosis samples. Increased IR affects cell cycle regulators at diagnosis and splicing regulators at remission. We show that aberrant splicing in CML is associated with reduced expression of specific splicing factors, histone modifications and reduced DNA methylation. Our results provide novel insights into the changing transcriptomic and epigenomic landscapes of CML patients during remission. The conceptually unanticipated observation of widespread aberrant alternative splicing after remission induction warrants further exploration. These results have broad implications for studying CML relapse and treating minimal residual disease

Comprehensive evaluation of targeted multiplex bisulphite PCR sequencing for validation of DNA methylation biomarker panels

Background: DNA methylation is a well-studied epigenetic mark that is frequently altered in diseases such as cancer, where specific changes are known to reflect the type and severity of the disease. Therefore, there is a growing interest in assessing the clinical utility of DNA methylation as a biomarker for diagnosing disease and guiding treatment. The development of an accurate loci-specific methylation assay, suitable for use on low-input clinical material, is crucial for advancing DNA methylation biomarkers into a clinical setting. A targeted multiplex bisulphite PCR sequencing approach meets these needs by allowing multiple DNA methylated regions to be interrogated simultaneously in one experiment on limited clinical material. Results: Here, we provide an updated protocol and recommendations for multiplex bisulphite PCR sequencing (MBPS) assays for target DNA methylation analysis. We describe additional steps to improve performance and reliability: (1) pre-sequencing PCR optimisation which includes assessing the optimal PCR cycling temperature and primer concentration and (2) post-sequencing PCR optimisation to achieve uniform coverage of each amplicon. We use a gradient of methylated controls to demonstrate how PCR bias can be assessed and corrected. Methylated controls also allow assessment of the sensitivity of methylation detection for each amplicon. Here, we show that the MBPS assay can amplify as little as 0.625 ng starting DNA and can detect methylation differences of 1% with a sequencing coverage of 1000 reads. Furthermore, the multiplex bisulphite PCR assay can comprehensively interrogate multiple regions on 1-5 ng of formalin-fixed paraffin-embedded DNA or circulating cell-free DNA. Conclusions: The MBPS assay is a valuable approach for assessing methylated DNA regions in clinical samples with limited material. The optimisation and additional quality control steps described here improve the performance and reliability of this method, advancing it towards potential clinical applications in biomarker studies

Replication timing and epigenome remodelling are associated with the nature of chromosomal rearrangements in cancer

The connection between DNA replication timing and changes that occur to the epigenome in cancer are still poorly understood. Here, the authors perform Repli-Seq and integrated epigenome analyses and find that genomic regions that undergo long-range epigenetic deregulation in prostate cancer also show concordant differences in replication timing

DNA Hypermethylation Encroachment at CpG Island Borders in Cancer Is Predisposed by H3K4 Monomethylation Patterns

Promoter CpG islands are typically unmethylated in normal cells, but in cancer a proportion are subject to hypermethylation. Using methylome sequencing we identified CpG islands that display partial methylation encroachment across the 5′ or 3′ CpG island borders. CpG island methylation encroachment is widespread in prostate and breast cancer and commonly associates with gene suppression. We show that the pattern of H3K4me1 at CpG island borders in normal cells predicts the different modes of cancer CpG island hypermethylation. Notably, genetic manipulation of Kmt2d results in concordant alterations in H3K4me1 levels and CpG island border DNA methylation encroachment. Our findings suggest a role for H3K4me1 in the demarcation of CpG island methylation borders in normal cells, which become eroded in cancer.We thank the Wysocka Laboratory for the generous gift of wild-type and mutant mESCs (Dorighi et al., 2017) and Roger Daly for financial support of prostate cancer WGBS (Cancer Institute NSW, Australia, program grant).The Bill and Patricia Ritchie Foundation, Australia (C.C.G.); UNSW Sydney University International Postgraduate Award (UIPA), Australia (K.S.

Widespread Aberrant Alternative Splicing despite Molecular Remission in Chronic Myeloid Leukaemia Patients

Vast transcriptomics and epigenomics changes are characteristic of human cancers, including leukaemia. At remission, we assume that these changes normalise so that omics-profiles resemble those of healthy individuals. However, an in-depth transcriptomic and epigenomic analysis of cancer remission has not been undertaken. A striking exemplar of targeted remission induction occurs in chronic myeloid leukaemia (CML) following tyrosine kinase inhibitor (TKI) therapy. Using RNA sequencing and whole-genome bisulfite sequencing, we profiled samples from chronic-phase CML patients at diagnosis and remission and compared these to healthy donors. Remarkably, our analyses revealed that abnormal splicing distinguishes remission samples from normal controls. This phenomenon is independent of the TKI drug used and in striking contrast to the normalisation of gene expression and DNA methylation patterns. Most remarkable are the high intron retention (IR) levels that even exceed those observed in the diagnosis samples. Increased IR affects cell cycle regulators at diagnosis and splicing regulators at remission. We show that aberrant splicing in CML is associated with reduced expression of specific splicing factors, histone modifications and reduced DNA methylation. Our results provide novel insights into the changing transcriptomic and epigenomic landscapes of CML patients during remission. The conceptually unanticipated observation of widespread aberrant alternative splicing after remission induction warrants further exploration. These results have broad implications for studying CML relapse and treating minimal residual disease

MOESM1 of Guidelines for whole genome bisulphite sequencing of intact and FFPET DNA on the Illumina HiSeq X Ten

Additional file 1: Table 1. Comparison of number of SNPs called in both WGBS and spike-in WGS data at 13x and 30x coverage. Table 2. Comparison of number of SNPs concordant in spike-in WGS data with WGS-Gold Standard at 30x coverage. Table 3. Comparison of number of SNPs concordant in WGBS data with WGS-Gold Standard at 30x coverage. Table 4. Percentage of SNPs observed across different genomic contexts for WGS-GS and WGBS

MOESM2 of Guidelines for whole genome bisulphite sequencing of intact and FFPET DNA on the Illumina HiSeq X Ten

Additional file 2. Figure 1: Box plot showing the difference in coverage across CpG islands, CpG shores and other regions of the genome for each of the five library preparation methods compared. Figure 2 a, b Two representative examples of regions showing SNP in both the WGS and WGBS data of the same clinical sample. Figure 3 a Bar plot showing the percentage of SNPs from WGBS concordant in WGS-GS at ~ 26× coverage and the percentage of SNPs from spike-in WGS concordant in WGS-GS at 30× coverage. b A representative Venn diagram for one prostate cancer sample, 2ab showing the number of SNPs concordant at 26× coverage for WGBS and 30× coverage for spike-in WGS when compared with WGS-GS data. Figure 4 a, Plot showing the distribution of normalised frequency of number of SNPs called across regions of the genome with varying levels of GC content for WGBS and WGS-GS. b Plot showing the distribution of normalised frequency of number of SNPs called across regions of the genome with varying levels of methylation ratio of CpG sites nearest to a SNP within 50 bp. Figure 5 a, b Box plot showing the coverage distribution across exons, intergenic regions, introns, promoter regions and repeat regions of the genome for a cell line sequenced on one lane of HiSeq X Ten (a) and HiSeq 2500 (b). Figure 6 a, b Two examples showing the difference in distribution of reads for a FFPET library obtained from the TruMethyl WG method and Accel-NGS Methyl-Seq method across a CpG island. Figure 7 a, b Summary of workflow for achieving optimal coverage on the HiSeq X Ten for intact genomic DNA and FFPET DNA