159 Refining TCR clonotype identification with long-read sequencing technique

• DOI: 10.1136/jitc-2024-sitc2024.0159
• Publication Date: 2024-11-05T14:58:54Z
• Authors: Nicholas Da Zhi Ang; Wei Lin Tang; Javian Zheng Han Ng; Wendy Lee; Solomonraj Wilson; Ser Mei Koh; Menaka Priyadhars...; Xiaoyu Liu; Bernett Lee; Olaf Rotzchke; Mai Chan Lau
• Keywords: Refining (metallurgy); Identification

<h3>Background</h3> Beyond conventional short-read Next-Generation Sequencing (NGS), long-read sequencing technology, like Oxford Nanopore Technologies (ONT),¹ enhance coverage and resolution of genomics fragments, crucial for detecting T Cell Receptors (TCRs) known their variability from VDJ recombination.² Short-read NGS sequences 3' or 5' end the cDNA template, sequencing, adapters/barcodes near constant regions hinder capturing variable regions. Long-read captures entire TCR regions, providing better on complete CDR3.³ However, long-reads have higher error rates,⁴ requiring a robust bioinformatic pipeline.^{5 6} We aim to improve accuracy reliability repertoire reconstruction, enhancing clonotype identification T-cell vaccine development. <h3>Methods</h3> Human PBMCs were processed full-length libraries using Chromium Next GEM Single v2 (10xGenomics) sequenced illumina HiSeq X, producing single-cell (scTCR) dataset (figure 1A). A separate aliquot was (ONT) Ligation Kit V14 PromethION Flow 1A), with dorado Duplex base-calling, scTCR dataset. cell barcodes identified following adaptor 2A). In dataset, adaptors removed, located TSO sequence. Extracted validated against whitelist,⁷ visualized VennDiagram R package. Reads segregated by unique identifying cell-specific alpha/beta chains. Hierarchical clustering performed Mash distances⁸ Ward's Linkage. clusters verified MiXCR-align.⁹ errors assessed MiXCR reference minimap2,¹⁰ IGV. <h3>Results</h3> introduce position-based barcode approach 2A), aid determination. Analysis shows that few matched 10x whitelist, while majority did not 2B). Over half dataset's also failed match, suggesting correction is needed. Using reads selected cell, revealed two main sequence 3A); one enriched in alpha, other beta, 3B - top; genes detailed at bottom). IGV visualization confirmed consistent basecalls 3C). <h3>Conclusions</h3> presented workflow refines analysis improving validity enabling precise reconstruction through sequencing. Further refinement UMI could analysis. This adaptable single-cell/spatial technologies. <h3>Acknowledgements</h3> work supported Bioinformatics Institute (BII), Singapore Immunology Network (SIgN), Agency Science, Technology Research (A*STAR). funded H22J1a0043 MOH-OFYIRG23jan-0021. <h3>References</h3> Lin B, Hui J, Mao H. technology its applications gene <i>Biosensors</i> 2021;<b>11</b>(7):214. https://doi.org/10.3390/bios11070214 Singh M, Al-Eryani G, Carswell S, Ferguson JM, Blackburn Barton K, Roden D, Luciani F, Giang Phan T, Junankar Jackson Goodnow CC, Smith MA, Swarbrick A. High-throughput targeted single reveals clonal transcriptional landscape lymphocytes. <i>Nature Communications</i> 2019;<b>10</b>(1):3120. https://doi.org/10.1038/s41467-019-11049-4 Mika Candéias SM, Badie C, Polanska J. (2022). Can we detect receptors RNA-Seq data?. In: Rojas I, Valenzuela O, Herrera LJ, Ortuño F. (eds) Biomedical Engineering. IWBBIO 2022. Lecture Notes Computer Science(), vol 13347. Springer, Cham. https://doi.org/10.1007/978-3-031-07802-6_38 Senol Cali Kim JS, Ghose Alkan Mutlu O. tools genome assembly: computational current state, bottlenecks future directions. <i>Briefings Bioinformatics</i> 2019;<b>20</b>(4):1542–1559. https://doi.org/10.1093/bib/bby017 Oehler JB, Wright H, Stark Z, Mallett AJ, Schmitz U. The application clinical settings. <i>Human Genomics</i>, 2023;<b>17</b>(1):73. https://doi.org/10.1186/s40246-023-00522-3 Gupta Witas R, Voigt A, Semenova Nguyen CQ. Single-cell receptors: perspective technological development translational application. <i>Advances Experimental medicine Biology</i> 2020;<b>1255</b>:29–50. https://doi.org/10.1007/978-981-15-4494-1_3 GENOMICS. What whitelist?. available at: https://kb.10xgenomics.com/hc/en-us/articles/115004506263-What-is-a-barcode-whitelist (Oct. 2023) Ondov BD, Treangen TJ, Melsted P.<i> et al</i>. Mash: fast metagenome distance estimation MinHash. <i>Genome Biol</i> 2016;<b>17</b>:132. https://doi.org/10.1186/s13059-016-0997-x Bolotin DA, Poslavsky Mitrophanov Shugay Mamedov IZ, Putintseva EV, Chudakov DM. MiXCR: software comprehensive adaptive immunity profiling.<i> Nat Methods</i> 2015 May;<b>12</b>(5):380-1. doi: 10.1038/nmeth.3364. PMID: 25924071. Heng Li, Minimap2: pairwise alignment nucleotide sequences. <i>Bioinformatics</i>, September 2018;<b>34</b>(18):3094–3100, https://doi.org/10.1093/bioinformatics/bty191