Once the sequencing was complete on the MinION flow cell, Nanopore MinKNOW sequencing software output FAST5 files were basecalled to FASTQ files and separated by barcode using Oxford Nanopore’s Guppy basecaller (version 2.3.5 or other where noted). The reads in the barcoded FASTQ files were aligned to human genome 19 using minimap2 version 2.17 (19 (link)) and resulting BAM files were sorted using SAMtools version 1.9 (20 (link)). Variant allele fraction (VAF) for each sample and gene of interest was then determined using Integrated Genome Viewer (IGV, version 2.61). VAF was calculated as mutant reads divided by the sum of mutant and wild type reads at that chromosomal location (see Supplemental Table S1 for chromosomal locations for SNPs of interest). When possible, up to three replicates were performed for sequencing. Reported VAF represents the mean of replicate values. Positive values are those with mean replicate values greater than our lower limit VAF (see Results ).
Minknow sequencing software
Manufactured by Oxford Nanopore
MinKNOW is the sequencing control software for Oxford Nanopore's DNA/RNA sequencing devices. It manages the sequencing run, collects and processes the raw nanopore signal data, and outputs sequence data.
Automatically generated - may contain errors
2 protocols using minknow sequencing software
1
Nanopore Sequencing Variant Detection Protocol
2
Nanopore sequencing of fungal pathogens
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The MinION sequencer (Oxford Nanopore Technologies, Oxford, UK) was used for sequencing the enriched libraries. Sequencing followed the manufacturer’s protocols for the Ligation sequencing kit (SQK-LSK109; Oxford Nanopore Technologies). The F. circinatum enriched library was sequenced on a MinION flow cell (R9.4.1, Oxford Nanopore Technologies) using MinKNOW Sequencing software (version 21.05.25, Oxford Nanopore Technologies). The native barcoding kit (EXP-NBD104, Oxford Nanopore Technologies) was used to barcode and pool the A. rolfsii and S. sclerotiorum enriched DNA libraries for sequencing on another flow cell. Basecalling was conducted using Guppy (version 3.2.2) after sequencing [93 (link)]. Raw reads from the pooled samples were demultiplexed, and adaptors were trimmed using Porechop (version 0.2.4) [94 ]. The quality of reads was assessed using NanoPlot (version 1.30.1) [95 ], followed by read quality filtering via Filtlong (version 0.2.0) [96 ]. Reads that are longer than 1 kb and with a quality score above 7 were kept [97 (link)].
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