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sqk-nbd114.96 protocol  (Oxford Nanopore)
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HLA typing workflow. (1) 100 ng gDNA is added to a PCR master mix and a two‐step PCR is performed. (2) The <t>amplicons</t> are End‐repaired and dA‐tailed. (3) Native barcodes from ONT are ligated onto the amplicons. (4) Samples are pooled and cleaned with 0.4× AMPure XP beads. (5) Sequencing adapters are ligated onto the barcoded amplicons and cleaned 0.4× AMPure XP beads. (6) The flow cell is prepared, and the sequencing library is loaded. (7) Sequencing is started, with a minimum QScore of 10 and demultiplexing on. (8) 1 fastq file per sample with a maximum of 4000 reads is analysed using NGSEngine‐Turbo. Created with BioRender.com.
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HLA typing workflow. (1) 100 ng gDNA is added to a PCR master mix and a two‐step PCR is performed. (2) The amplicons are End‐repaired and dA‐tailed. (3) Native barcodes from ONT are ligated onto the amplicons. (4) Samples are pooled and cleaned with 0.4× AMPure XP beads. (5) Sequencing adapters are ligated onto the barcoded amplicons and cleaned 0.4× AMPure XP beads. (6) The flow cell is prepared, and the sequencing library is loaded. (7) Sequencing is started, with a minimum QScore of 10 and demultiplexing on. (8) 1 fastq file per sample with a maximum of 4000 reads is analysed using NGSEngine‐Turbo. Created with BioRender.com.

Journal: Hla

Article Title: Cost‐Effective and Highly Scalable Typing of HLA Classes I and II Genes of up to 96 Individuals Using Nanopore Sequencing

doi: 10.1111/tan.70164

Figure Lengend Snippet: HLA typing workflow. (1) 100 ng gDNA is added to a PCR master mix and a two‐step PCR is performed. (2) The amplicons are End‐repaired and dA‐tailed. (3) Native barcodes from ONT are ligated onto the amplicons. (4) Samples are pooled and cleaned with 0.4× AMPure XP beads. (5) Sequencing adapters are ligated onto the barcoded amplicons and cleaned 0.4× AMPure XP beads. (6) The flow cell is prepared, and the sequencing library is loaded. (7) Sequencing is started, with a minimum QScore of 10 and demultiplexing on. (8) 1 fastq file per sample with a maximum of 4000 reads is analysed using NGSEngine‐Turbo. Created with BioRender.com.

Article Snippet: The PCR took around 2 h, after which the resulting amplicons had barcodes ligated onto them according to the SQK‐NBD114.96 protocol (Oxford Nanopore Technologies, Oxford, UK) with slight modifications, taking approximately an additional 2.5 h. All 96 samples were sequenced on the same MinION R10.4.1 flow cell.

Techniques: Immunopeptidomics, Sequencing