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

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          Abstract

          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.

          Supplementary Information

          The online version contains supplementary material available at 10.1186/s13059-020-02240-8.

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          Most cited references81

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          Trimmomatic: a flexible trimmer for Illumina sequence data

          Motivation: Although many next-generation sequencing (NGS) read preprocessing tools already existed, we could not find any tool or combination of tools that met our requirements in terms of flexibility, correct handling of paired-end data and high performance. We have developed Trimmomatic as a more flexible and efficient preprocessing tool, which could correctly handle paired-end data. Results: The value of NGS read preprocessing is demonstrated for both reference-based and reference-free tasks. Trimmomatic is shown to produce output that is at least competitive with, and in many cases superior to, that produced by other tools, in all scenarios tested. Availability and implementation: Trimmomatic is licensed under GPL V3. It is cross-platform (Java 1.5+ required) and available at http://www.usadellab.org/cms/index.php?page=trimmomatic Contact: usadel@bio1.rwth-aachen.de Supplementary information: Supplementary data are available at Bioinformatics online.
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            STAR: ultrafast universal RNA-seq aligner.

            Accurate alignment of high-throughput RNA-seq data is a challenging and yet unsolved problem because of the non-contiguous transcript structure, relatively short read lengths and constantly increasing throughput of the sequencing technologies. Currently available RNA-seq aligners suffer from high mapping error rates, low mapping speed, read length limitation and mapping biases. To align our large (>80 billon reads) ENCODE Transcriptome RNA-seq dataset, we developed the Spliced Transcripts Alignment to a Reference (STAR) software based on a previously undescribed RNA-seq alignment algorithm that uses sequential maximum mappable seed search in uncompressed suffix arrays followed by seed clustering and stitching procedure. STAR outperforms other aligners by a factor of >50 in mapping speed, aligning to the human genome 550 million 2 × 76 bp paired-end reads per hour on a modest 12-core server, while at the same time improving alignment sensitivity and precision. In addition to unbiased de novo detection of canonical junctions, STAR can discover non-canonical splices and chimeric (fusion) transcripts, and is also capable of mapping full-length RNA sequences. Using Roche 454 sequencing of reverse transcription polymerase chain reaction amplicons, we experimentally validated 1960 novel intergenic splice junctions with an 80-90% success rate, corroborating the high precision of the STAR mapping strategy. STAR is implemented as a standalone C++ code. STAR is free open source software distributed under GPLv3 license and can be downloaded from http://code.google.com/p/rna-star/.
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              Fast gapped-read alignment with Bowtie 2.

              As the rate of sequencing increases, greater throughput is demanded from read aligners. The full-text minute index is often used to make alignment very fast and memory-efficient, but the approach is ill-suited to finding longer, gapped alignments. Bowtie 2 combines the strengths of the full-text minute index with the flexibility and speed of hardware-accelerated dynamic programming algorithms to achieve a combination of high speed, sensitivity and accuracy.
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                Author and article information

                Contributors
                m.oka@ono.co.jp
                leahxuliu@gmail.com
                toshsuzu@east.ncc.go.jp
                toyoshik@east.ncc.go.jp
                hsakamot@ncc.go.jp
                hayato.u88101204@gmail.com
                acyshzw@pharm.kyoto-u.ac.jp
                ysuzuki@hgc.jp
                tnakatsu@east.ncc.go.jp
                yishiham@pharm.kyoto-u.ac.jp
                asuzuki@edu.k.u-tokyo.ac.jp
                mseki@edu.k.u-tokyo.ac.jp
                Journal
                Genome Biol
                Genome Biol
                Genome Biology
                BioMed Central (London )
                1474-7596
                1474-760X
                4 January 2021
                4 January 2021
                2021
                : 22
                : 9
                Affiliations
                [1 ]GRID grid.26999.3d, ISNI 0000 0001 2151 536X, Department of Computational Biology and Medical Sciences, , Graduate School of Frontier Sciences, The University of Tokyo, ; Chiba, Japan
                [2 ]GRID grid.459873.4, ISNI 0000 0004 0376 2510, Ono Pharmaceutical Co., Ltd., ; Ibaraki, Japan
                [3 ]GRID grid.264706.1, ISNI 0000 0000 9239 9995, General Medical Education and Research Center, , Teikyo University, ; Tokyo, Japan
                [4 ]GRID grid.272242.3, ISNI 0000 0001 2168 5385, Division of Cancer Immunotherapy, , Exploratory Oncology Research and Clinical Trial Center, National Cancer Center, ; Chiba, Japan
                [5 ]GRID grid.272242.3, ISNI 0000 0001 2168 5385, Department of Clinical Genomics, , National Cancer Center Research Institute, ; Tokyo, Japan
                [6 ]GRID grid.258799.8, ISNI 0000 0004 0372 2033, Department of Molecular and Cellular BioAnalysis, Graduate School of Pharmaceutical Sciences, , Kyoto University, ; Kyoto, Japan
                Article
                2240
                10.1186/s13059-020-02240-8
                7780684
                33397462
                6c6f81fb-6221-4d83-aa76-96d73c3b38dd
                © The Author(s) 2021

                Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

                History
                : 13 August 2020
                : 14 December 2020
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100001691, Japan Society for the Promotion of Science;
                Award ID: 16H06279
                Award ID: 19K16108
                Award ID: 19K16792
                Award Recipient :
                Funded by: National Cancer Center Research and Development Fund
                Award ID: 29-A-2
                Award Recipient :
                Categories
                Research
                Custom metadata
                © The Author(s) 2021

                Genetics
                minion,lung cancer,splicing isoform,neoantigen
                Genetics
                minion, lung cancer, splicing isoform, neoantigen

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