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  Indian J Med Microbiol
 

Figure 1: (a) Overview of workflow. The reagents and informatics methodology have been incorporated into the publicly available Oncomine TCR Beta-LR assay and accompanying Ion Reporter analysis software. (b) Method for library preparation and sequencing. Our strategy utilizes multiplex polymerase chain reaction (PCR) through framework 1 and constant gene primers followed by bidirectional next-generation sequencing (NGS) of amplicon libraries. (c) Error profile of the S5 530 chip. The average substitution error rate for the S5 530 chip was calculated by sequencing of the E. coli dh1b genome and mapping sequence reads to reference through the Torrent accuracy plugin for Torrent Suite. (d) Comparison of substitution error rates across three Illumina sequencers and the Ion Torrent S5 530 chip. To account for differences in reading lengths between the Illumina and Ion Torrent platforms, the error rate was calculated as the average over the bases 10-100 of the read. The first 10 base pairs were excluded given that alignment scores cannot accurately distinguish indel errors from substitution errors at the read ends. Error rates for the Illumina platform are taken from Schirmer et al., 2016.[21] (e) Strategy for identification of non-IMGT variable gene alleles. Bona fide novel alleles will present as systematic mismatches to IMGT across a plurality of clones, each possessing a distinct CDR3 nucleotide sequence. IGMT: ImMunoGeneTics; TRBV: T - cell receptor beta variable gene; FFPE: Formalin - fixed paraffin - embedded

Figure 1: (a) Overview of workflow. The reagents and informatics methodology have been incorporated into the publicly available Oncomine TCR Beta-LR assay and accompanying Ion Reporter analysis software. (b) Method for library preparation and sequencing. Our strategy utilizes multiplex polymerase chain reaction (PCR) through framework 1 and constant gene primers followed by bidirectional next-generation sequencing (NGS) of amplicon libraries. (c) Error profile of the S5 530 chip. The average substitution error rate for the S5 530 chip was calculated by sequencing of the <i>E. coli</i> dh1b genome and mapping sequence reads to reference through the Torrent accuracy plugin for Torrent Suite. (d) Comparison of substitution error rates across three Illumina sequencers and the Ion Torrent S5 530 chip. To account for differences in reading lengths between the Illumina and Ion Torrent platforms, the error rate was calculated as the average over the bases 10-100 of the read. The first 10 base pairs were excluded given that alignment scores cannot accurately distinguish indel errors from substitution errors at the read ends. Error rates for the Illumina platform are taken from Schirmer et al<i>.</i>, 2016.<sup>[21]</sup> (e) Strategy for identification of non-IMGT variable gene alleles. Bona fide novel alleles will present as systematic mismatches to IMGT across a plurality of clones, each possessing a distinct CDR3 nucleotide sequence. IGMT: ImMunoGeneTics; TRBV: T - cell receptor beta variable gene; FFPE: Formalin - fixed paraffin - embedded