Flo min106d r9.4.1 flow cell

RPRD: To fully characterize samples with CNVs, XL-PCR amplicons encompassing the duplicated and/or nonduplicated gene copies were generated and subjected to single molecule sequencing using Nanopore as previously described [36 (link),39 (link)]. Primers and PCR conditions used to generate the amplicons are shown in Table S2 and are as previously described [33 (link)]. Libraries were prepared using the Ligation Sequencing DNA Kit (SQK-LSK109) and Native Barcoding Kit EXP-NBD104 (Oxford Nanopore Technologies, Oxford, UK) per manufacturer recommendations. Barcoded libraries were pooled and sequenced on the MinION Sequencing instrument with a FLO-MIN106D (R9.4.1) flow cell (Oxford Nanopore Technologies, Oxford, UK) as prescribed to a minimum read depth of 10,000×). Reads were filtered for qscore >10 and base-called with MinKNOW (v21.06.13). FASTQ files were mapped to GRCh38 (NC_000022.11) using Minimap2 [40 (link)], and variant calling was performed using Nanopolish [41 (link)]. Thresholds were set as recommended (0.2). Alignments were viewed using the Integrative Genomics Viewer v.2.4.1 (IGV, Broad 153 Institute, Boston, MA, USA) aligning to the human GRCh38 reference genome to confirm genotype calls [42 (link)].

The genomes of S. cerevisiae WY3711 and A62 were sequenced using an Oxford Nanopore MinION. Genomic DNA was extracted with a YeaStar Genomic DNA kit (Zymo Research, Irvine, CA, USA) and then barcoded with the Native Barcoding kit (EXP-NBD104; Oxford Nanopore Technology, United Kingdom). A 1D sequencing library was then prepared from the barcoded DNA using the Ligation Sequencing Kit (SQK-LSK109; Oxford Nanopore Technology, United Kingdom). The library was sequenced on a FLO-MIN106D (R9.4.1) flow cell with a MinION device (Oxford Nanopore Technology, United Kingdom). The raw fast5 files were basecalled using the GPU-version of Guppy (version 2.3.7; using the supplied dna_r9.4.1_450bps_flipflop.cfg configuration). The basecalled reads were demultiplexed with qcat (version 1.0.1; available from https://github.com/nanoporetech/qcat), and filtered to a minimum length of 1000 bp and average read quality score of 10 using NanoFilt (version 2.2.0; available from https://github.com/wdecoster/nanofilt). This resulted in a total of 1.3 Gbp of sequence for S. cerevisiae WY3711 (coverage of 108×) and 1.9 Gbp of sequence for S. cerevisiae A81062 (coverage of 155×).