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.. _preparation:
Data preparation from raw reads
===================================
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 <https://nanoporetech.com>`_::
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 <https://nanopolish.readthedocs.io/en/latest/quickstart_eventalign.html>`_::
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>
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