Long-read whole-genome methylation patterning using enzymatic base conversion and nanopore sequencing

Sakamoto, Yoshitaka; Zaha, Suzuko; Nagasawa, Satoi; Miyake, Shuhei; Kojima, Yasuyuki; Suzuki, Ayako; Suzuki, Yutaka; Seki, Masahide

doi:10.1093/nar/gkab397

Cited by 32 publications

(26 citation statements)

References 50 publications

(74 reference statements)

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“…DNA methylation analysis. CpG methylation sites were called from fast5 data, and we calculated their methylation frequency using nanopolish (version 0.13.2) 25,64 under the settings as shown in Supplementary Methods. Using the haplotype-tagged PromethION reads from "WhatsHap haplotag," we classified the reads into two haplotypes and calculated the methylation frequencies in each haplotype category using the given script in nanopolish.…”

Section: Methodsmentioning

confidence: 99%

Phasing analysis of lung cancer genomes using a long read sequencer

et al. 2022

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Chromosomal backgrounds of cancerous mutations still remain elusive. Here, we conduct the phasing analysis of non-small cell lung cancer specimens of 20 Japanese patients. By the combinatory use of short and long read sequencing data, we obtain long phased blocks of 834 kb in N50 length with >99% concordance rate. By analyzing the obtained phasing information, we reveal that several cancer genomes harbor regions in which mutations are unevenly distributed to either of two haplotypes. Large-scale chromosomal rearrangement events, which resemble chromothripsis events but have smaller scales, occur on only one chromosome, and these events account for the observed biased distributions. Interestingly, the events are characteristic of EGFR mutation-positive lung adenocarcinomas. Further integration of long read epigenomic and transcriptomic data reveal that haploid chromosomes are not always at equivalent transcriptomic/epigenomic conditions. Distinct chromosomal backgrounds are responsible for later cancerous aberrations in a haplotype-specific manner.

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Section: Methodsmentioning

confidence: 99%

Phasing analysis of lung cancer genomes using a long read sequencer

et al. 2022

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“…Hence, there is no published guideline and systematic comparison of all current DNA methylation-calling tools for nanopore sequencing using natural human DNA [44], especially at the whole-epigenome scale. Recently, research with the combination of bisulfite-free enzymatic base conversion and nanopore sequencing [45][46][47] enabled high accuracy and potency in long-range epigenetic phasing. Together, these studies opened up new and orthogonal approaches to uncover the long-range coordination of epigenetic marks at single-molecule, single-base resolution.…”

Section: Introductionmentioning

confidence: 99%

DNA methylation-calling tools for Oxford Nanopore sequencing: a survey and human epigenome-wide evaluation

et al. 2021

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Background Nanopore long-read sequencing technology greatly expands the capacity of long-range, single-molecule DNA-modification detection. A growing number of analytical tools have been developed to detect DNA methylation from nanopore sequencing reads. Here, we assess the performance of different methylation-calling tools to provide a systematic evaluation to guide researchers performing human epigenome-wide studies. Results We compare seven analytic tools for detecting DNA methylation from nanopore long-read sequencing data generated from human natural DNA at a whole-genome scale. We evaluate the per-read and per-site performance of CpG methylation prediction across different genomic contexts, CpG site coverage, and computational resources consumed by each tool. The seven tools exhibit different performances across the evaluation criteria. We show that the methylation prediction at regions with discordant DNA methylation patterns, intergenic regions, low CG density regions, and repetitive regions show room for improvement across all tools. Furthermore, we demonstrate that 5hmC levels at least partly contribute to the discrepancy between bisulfite and nanopore sequencing. Lastly, we provide an online DNA methylation database (https://nanome.jax.org) to display the DNA methylation levels detected by nanopore sequencing and bisulfite sequencing data across different genomic contexts. Conclusions Our study is the first systematic benchmark of computational methods for detection of mammalian whole-genome DNA modifications in nanopore sequencing. We provide a broad foundation for cross-platform standardization and an evaluation of analytical tools designed for genome-scale modified base detection using nanopore sequencing.

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“…Experimental procedures were also approved under the same approval number. Case A is derived from the same specimens as a tumor tissue of Case 8 used in our previous study 45 . We subjected whole tumor tissues to dissection of a few mm in thickness without excluding a specific area to prepare single cells and semibulks.…”

Section: Methodsmentioning

confidence: 99%

Semibulk RNA-seq analysis as a convenient method for measuring gene expression statuses in a local cellular environment

Muto

Tsuchiya

Kim

et al. 2022

Sci Rep

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When biologically interpretation of the data obtained from the single-cell RNA sequencing (scRNA-seq) analysis is attempted, additional information on the location of the single cells, behavior of the surrounding cells, and the microenvironment they generate, would be very important. We developed an inexpensive, high throughput application while preserving spatial organization, named “semibulk RNA-seq” (sbRNA-seq). We utilized a microfluidic device specifically designed for the experiments to encapsulate both a barcoded bead and a cell aggregate (a semibulk) into a single droplet. Using sbRNA-seq, we firstly analyzed mouse kidney specimens. In the mouse model, we could associate the pathological information with the gene expression information. We validated the results using spatial transcriptome analysis and found them highly consistent. When we applied the sbRNA-seq analysis to the human breast cancer specimens, we identified spatial interactions between a particular population of immune cells and that of cancer-associated fibroblast cells, which were not precisely represented solely by the single-cell analysis. Semibulk analysis may provide a convenient and versatile method, compared to a standard spatial transcriptome sequencing platform, to associate spatial information with transcriptome information.

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Long-read whole-genome methylation patterning using enzymatic base conversion and nanopore sequencing

Cited by 32 publications

References 50 publications

Phasing analysis of lung cancer genomes using a long read sequencer

Phasing analysis of lung cancer genomes using a long read sequencer

DNA methylation-calling tools for Oxford Nanopore sequencing: a survey and human epigenome-wide evaluation

Semibulk RNA-seq analysis as a convenient method for measuring gene expression statuses in a local cellular environment

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