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This is a general protocol for monitoring and dealing with a MinION sequencing run.
Programs used: albacore (https://github.com/dvera/albacore), Porechop (https://github.com/rrwick/Porechop), ABruijn (https://github.com/fenderglass/ABruijn), Canu (http://canu.readthedocs.io/en/latest/quick-start.html), smartdenovo (https://github.com/ruanjue/smartdenovo), miniasm (https://github.com/lh3/miniasm), NanoPlot (https://github.com/wdecoster/NanoPlot)
Make sure there is enough disk space for the sequencing data to be stored.
Once you've loaded your flow cell and hit execute, keep an eye on the MinKNOW interface. Things you need to look out for:
- The muxing steps show similar pore availability as when the flow cell was QCed.
- The active pore to in-strand pore ratio should preferably be 1:1.
- The fragment distribution shows sizes you'd expect to see (if your DNA was fragmented to 8kb, you should see most of your “reads” in this range and some below this size).
- The biggest amount of data is produced during the first 12 hours. If there isn't much more data generated after that, you might want to RESTART the run. If that doesn't produce more data, STOP the run before it goes to completion (a run is usually completed in 48 hours) and wash the flow cell and store it for later use.
Number of "events" x 1.8 = Number of basepairs
Transfer the raw fast5 files using an FTP client such as FileZilla (https://filezilla-project.org/) to your Perun account. This might take up to a day.
Basecalling
Here is an example of a script for albacore:
#!/bin/bash #$ -S /bin/bash . /etc/profile #$ -cwd #$ -pe threaded 20 source /scratch2/software/Python-3.6.0/set-path read_fast5_basecaller.py --input /scratch2/sarahshah/MINION_RAW_DATA/Roach/20171103_1655_RoachBlasto --worker_threads 20 --save_path /scratch2/sarahshah/MINION_RAW_DATA/Roach/reads --flo wcell FLO-MIN106 --kit SQK-LSK108 --recursive --files_per_batch_folder 0 --output_format fastq --reads_per_fastq_batch 9999999999999 read_fast5_basecaller.py --input /scratch2/sarahshah/MINION_RAW_DATA/Roach/20171103_1702_RoachBlasto --worker_threads 20 --save_path /scratch2/sarahshah/MINION_RAW_DATA/Roach/reads --flo wcell FLO-MIN106 --kit SQK-LSK108 --recursive --files_per_batch_folder 0 --output_format fastq --reads_per_fastq_batch 9999999999999
The “read_fast5_basecaller.py” line should be repeated for each input folder.
Adapter Trimming using Porechop
Porechop trims adapters from ends or inside of reads. Below is an example of a shell script:
#!/bin/bash #$ -S /bin/bash . /etc/profile #$ -cwd #$ -pe threaded 16 export PATH=/scratch2/software/Python-3.6.0/bin:/scratch2/software/gcc-6.3.0/bin:$PATH export LD_LIBRARY_PATH=/scratch2/software/Python-3.6.0/lib:/scratch2/software/gcc-6.3.0/lib64:$LD_LIBRARY_PATH unset PYTHONPATH porechop -i /scratch2/ginaf/Phaeo_minion/cut_reads/Phaeo_MERGED_uncut.fastq -o /scratch2/ginaf/Phaeo_minion/cut_reads/Phaeo_MERGED_CHOPPED.fastq --threads 16 --verbosity 2
Check your trimmed fastq quality using NanoStat/Plot