Biology subjects
Publications and source records attributed to Ishii, J..
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Mass-based fingerprinting can characterize unknown strains, however expansion of these methods to predict specific gene functions is lacking. Therefore, rapid mass fingerprinting was developed to functionally profile a comprehensive yeast knockout library. Matrix assisted laser desorption ionization (MALDI)-time of flight (TOF) mass fingerprints of 3,238 Saccharomyces cerevisiae knockouts were digitized for correlation with gene ontology (GO) annotations. Random forests and support vector machine (SVM) algorithms precisely assigned GO accessions with AUC scores all above 0.83. SVM was the best predictor with average true positive and true negative rates of 0.975 and 0.991, respectively. The SVM model suggested new functions for 28 uncharacterized yeast genes. Metabolomics analysis of two knockouts (YDR215C and YLR122C) of uncharacterized genes predicted to be involved in methylation-related metabolism, showed altered intracellular contents of methionine-related metabolites. Increased S-adenosylmethionine in YDR215C highlights potential for enhancement of methylation pathways. These results demonstrate that MALDI-TOF fingerprints can be rapidly digitized, resulting in datasets that enable prediction of microbial genotypes and even the function of specific genes. This fingerprinting method can inform optimal bioproduction chassis selection.
Cytosine modifications in DNA such as 5-methylcytosine (5mC) underlie a broad range of developmental processes, maintain cellular lineage specification, and can define or stratify cancer and other diseases. However, the wide variety of approaches available to interrogate these modifications has created a need for harmonized materials, methods, and rigorous benchmarking to improve genome-wide methylome sequencing applications in clinical and basic research. Here, we present a multi-platform assessment and a global resource for epigenetics research from the FDAs Epigenomics Quality Control (EpiQC) Group. The study design leverages seven human cell lines that are designated as reference materials and publicly available from the National Institute of Standards and Technology (NIST) and Genome in a Bottle (GIAB) consortium. These samples were subject to a variety of genome-wide methylation interrogation approaches across six independent laboratories, with a primary focus was on 5-methylcytosine modifications. Each sample was processed in two or more technical replicates by three whole-genome bisulfite sequencing (WGBS) protocols (TruSeq DNA methylation, Accel-NGS MethylSeq, and SPLAT), oxidative bisulfite sequencing (TrueMethyl), one enzymatic deamination method (EMseq), targeted methylation sequencing (Illumina Methyl Capture EPIC), and single-molecule long-read nanopore sequencing from Oxford Nanopore Technologies. After rigorous quality assessment and comparison to Illumina EPIC methylation microarrays and testing on a range of algorithms (Bismark, BitmapperBS, BWAMeth, and GemBS), we found overall high concordance between assays (R=0.87-R0.93), differences in efficency of read mapping and CpG capture and coverage, and platform performance. The data provided herein can guide continued used of these reference materials in epigenomics assays, as well as provide best practices for epigenomics research and experimental design in future studies.
Some cancer cells lengthen their telomeres by alternative lengthening of telomeres (ALT); these are referred to as ALT cancer cells and do not express telomerase. The ALT mechanism involves the elongation of telomeric DNA repeats by homologous recombination. In interphase nuclei of ALT cancer cells, giant telomeres can be specifically observed by fluorescence in situ hybridization (FISH) to detect telomeric repeats, and they are co-localized with promyelocytic-leukemia nuclear-bodies (PML-NBs). However, it how large telomeres specific to ALT and how they form a structural relation with PML-NBs. We refer to giant telomeres specific to interphase ALT cancer cells giant-telomeric nuclear-bodies (GT-NBs). To quantitatively define GT-NBs, we performed telomeric FISH of both interphase nuclei and metaphase chromosomes and analyzed telomere sizes by integrated FISH signals. The distributions of telomere sizes in telomerase-positive cells were similar in interphase nuclei and chromosomes. However, the distribution of telomere sizes in ALT cancer cells differed between interphase nuclei and chromosomes. Giant telomeres that were larger than those at chromosomal ends were only observed in interphase nuclei of ALT cancer cells. Accordingly, GT-NBs could be quantitatively defined as larger than the maximum size of telomeres at chromosomal ends. Furthermore, ALT cancer cells demonstrated fewer telomeric signals in interphase nuclei than in chromosomes. These findings indicate that GT-NBs could be formed by the aggregation of two or more telomeres at chromosomal ends. Furthermore, super-resolution microscopy showed that GT-NBs contain one or two PML-NBs. GT-NBs are considered aggregates of telomeres and could contain multiple sites of homologous recombination accompanied by PML-NBs. These findings may contribute to the development of therapeutic approaches for ALT cancer. (251 words)
The Illumina HiSeq X platform has helped to reduce the cost of whole genome sequencing substantially, but its application for bisulphite sequencing is not straightforward. We describe the optimization of a library preparation and sequencing approach that maximizes the yield and quality of sequencing, and the elimination of a previously unrecognized artefact affecting several percent of bisulphite sequencing reads.