Long-read sequencing for non-small-cell lung cancer genomes

Sakamoto, Yoshitaka; Xu, Lai; Seki, Masahide; Yokoyama, Toshiyuki; Kasahara, Masahiro; Kashima, Yukie; Ohashi, Akihiro; Shimada, Yôko; Motoi, Noriko; Tsuchihara, Katsuya; Kobayashi, Susumu; Shiraishi, Yuichi; Suzuki, Ayako; Suzuki, Yutaka

doi:10.1101/gr.261941.120

Cited by 34 publications

(35 citation statements)

References 62 publications

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“…For the purpose of long-read sequencing, we employed a nanopore sequencer. A nanopore sequencer can potentially read DNA molecules that are longer than 100 kb ( 10 ). It has also been reported that modifications in DNA and RNA can be detected from the electric signal patterns observed for mC of DNA and RNA, hmC of DNA, and unmethylated C ( 11 , 12 ).…”

Section: Introductionmentioning

confidence: 99%

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

Sakamoto

Zaha

Nagasawa

et al. 2021

Nucleic Acids Research

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Long-read whole-genome sequencing analysis of DNA methylation would provide useful information on the chromosomal context of gene expression regulation. Here we describe the development of a method that improves the read length generated by using the bisulfite-sequencing-based approach. In this method, we combined recently developed enzymatic base conversion, where an unmethylated cytosine (C) should be converted to thymine (T), with nanopore sequencing. After methylation-sensitive base conversion, the sequencing library was constructed using long-range polymerase chain reaction. This type of analysis is possible using a minimum of 1 ng genomic DNA, and an N50 read length of 3.4–7.6 kb is achieved. To analyze the produced data, which contained a substantial number of base mismatches due to sequence conversion and an inaccurate base read of the nanopore sequencing, a new analytical pipeline was constructed. To demonstrate the performance of long-read methylation sequencing, breast cancer cell lines and clinical specimens were subjected to analysis, which revealed the chromosomal methylation context of key cancer-related genes, allele-specific methylated genes, and repetitive or deletion regions. This method should convert the intractable specimens for which the amount of available genomic DNA is limited to the tractable targets.

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Section: Introductionmentioning

confidence: 99%

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

Sakamoto

Zaha

Nagasawa

et al. 2021

Nucleic Acids Research

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“…In the past, the advantages of long-reads and real-time sequencing have made Nanopore sequencing an effective tool to detect genomic and genetics aberrations such as DNA structural variants and RNA alternative splicing events [53]. Nanopore sequencing have demonstrated its powerful capability of detecting structural variation in lung cancer [54, 55], leukemia [56], and neuron disorder [57–59], and it has been applied to clinical samples for molecular etiology or diagnosis of genomic variants relevant disease [57–62]. Meanwhile, Nanopore sequencing of splicing changes has been utilized in cancer research such as breast cancer [63], leukemia [64, 65], and brain tumor [66].…”

Section: Discussionmentioning

confidence: 99%

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

Liu

Rosikiewicz

Pan

et al. 2021

Preprint

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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 actively developed to detect DNA methylation from Nanopore sequencing reads. Here, we examine the performance of different methylation calling tools to provide a systematic evaluation to guide practitioners for human epigenome-wide research. Results: We compare five analytic frameworks for detecting DNA modification from Nanopore long-read sequencing data. We evaluate the association between genomic context, CpG methylation-detection accuracy, CpG sites coverage, and running time using Nanopore sequencing data from natural human DNA. Furthermore, we provide an online DNA methylation database (https://nanome.jax.org) with which to display genomic regions that exhibit differences in DNA-modification detection power among different methylation calling algorithms for nanopore sequencing data. Conclusions: Our study is the first benchmark of computational methods for mammalian whole genome DNA-modification detection 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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“…Base calling of the FAST5 data from MinION was performed using Albacore 2.2.7 and Guppy v3.3.0, then was converted into FASTQ files. Only 1D reads for the following analyses [ 62 ]. Reads were aligned to the reference human genome, GRCh38, using Minimap2 (v2.2.14) [ 33 ].…”

Section: Methodsmentioning

confidence: 99%

Aberrant splicing isoforms detected by full-length transcriptome sequencing as transcripts of potential neoantigens in non-small cell lung cancer

Oka

Suzuki

et al. 2021

Genome Biol

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Background Long-read sequencing of full-length cDNAs enables the detection of structures of aberrant splicing isoforms in cancer cells. These isoforms are occasionally translated, presented by HLA molecules, and recognized as neoantigens. This study used a long-read sequencer (MinION) to construct a comprehensive catalog of aberrant splicing isoforms in non-small-cell lung cancers, by which novel isoforms and potential neoantigens are identified. Results Full-length cDNA sequencing is performed using 22 cell lines, and a total of 2021 novel splicing isoforms are identified. The protein expression of some of these isoforms is then validated by proteome analysis. Ablations of a nonsense-mediated mRNA decay (NMD) factor, UPF1, and a splicing factor, SF3B1, are found to increase the proportion of aberrant transcripts. NetMHC evaluation of the binding affinities to each type of HLA molecule reveals that some of the isoforms potentially generate neoantigen candidates. We also identify aberrant splicing isoforms in seven non-small-cell lung cancer specimens. An enzyme-linked immune absorbent spot assay indicates that approximately half the peptide candidates have the potential to activate T cell responses through their interaction with HLA molecules. Finally, we estimate the number of isoforms in The Cancer Genome Atlas (TCGA) datasets by referring to the constructed catalog and found that disruption of NMD factors is significantly correlated with the number of splicing isoforms found in the TCGA-Lung Adenocarcinoma data collection. Conclusions Our results indicate that long-read sequencing of full-length cDNAs is essential for the precise identification of aberrant transcript structures in cancer cells.

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Long-read sequencing for non-small-cell lung cancer genomes

Cited by 34 publications

References 62 publications

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

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

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

Aberrant splicing isoforms detected by full-length transcriptome sequencing as transcripts of potential neoantigens in non-small cell lung cancer

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