Data preparation from raw readsΒΆ

  1. After obtaining fast5 files, the first step is to basecall them. Below is an example script to run Guppy basecaller. You can find more detail about basecalling at Oxford nanopore Technologies:

    guppy_basecaller -i </PATH/TO/FAST5> -s </PATH/TO/FASTQ> --flowcell <FLOWCELL_ID> --kit <KIT_ID> --device auto -q 0 -r
  2. Align to transcriptome:

    minimap2 -ax map-ont -uf -t 3 --secondary=no <MMI> <PATH/TO/FASTQ.GZ> > <PATH/TO/SAM> 2>> <PATH/TO/SAM_LOG>
    samtools view -Sb <PATH/TO/SAM> | samtools sort -o <PATH/TO/BAM> - &>> <PATH/TO/BAM_LOG>
    samtools index <PATH/TO/BAM> &>> <PATH/TO/BAM_INDEX_LOG>
  3. Resquiggle using nanopolish eventalign:

    nanopolish index -d <PATH/TO/FAST5_DIR> <PATH/TO/FASTQ_FILE>
    nanopolish eventalign --reads <PATH/TO/FASTQ_FILE> \
    --bam <PATH/TO/BAM_FILE> \
    --genome <PATH/TO/FASTA_FILE \
    --signal-index \
    --scale-events \
    --summary <PATH/TO/summary.txt> \
    --threads 32 > <PATH/TO/eventalign.txt>