Editing Nanopore RNA Sequencing Protocol (section)

====Guppy====

We’re going to install ''Guppy'' directly from ''Oxford Nanopore Technologies'', hereinthereafter simply ''ONT''. Since ''EpiNano'' models were basecalled with ''Guppy 3.1.5'', you will want to install and use the same version for ''EpiNano-SVM'', but note that this version has some requirements and limitations (see the below warning note). Additionally, ''Guppy 3.1.5'' is incredibly slow since it does not utilize a CUDA GPU, and so your analyses will be slower with ''Guppy 3.1.5''. For this protocol, I will install the latest ''Guppy (6.4.2)'' and use ''m6ANet'' for m6A detection.

{{warning|'''CRITICALLY IMPORTANT:''' ''Guppy 3.1.5'', the version for which the default models for ''EpiNano-SVM'' were based off of, utilizes old 32-bit libraries which are unavailable and no longer supported on 64-bit Linux. Therefore, if you plan to use it, you need to also copy these libraries to your <code>/usr/lib</code> folder for it to work. These library files are available [https://ftp5.gwdg.de/pub/linux/archlinux/community/os/x86_64/libidn11-1.33-2-x86_64.pkg.tar.zst here]. Additionally, ''Guppy 3.1.5'' is longer compatible with newly acquired <code>.fast5</code> data using MinKNOW 21.10+. I will use a newer version of ''Guppy'' and ''m6ANet'' as a result, but I kept the code on how to use ''EpiNano-SVM'' (and ''EpiNano-Error'' for that matter) below in case you’re running older data through ''Guppy 3.1.5'', downgraded your ''MinKNOW'' software, or found newer RNA models.}}

<code>Bash</code>
<syntaxhighlight lang="Bash">
GUPPYVER="6.4.2"
wget https://mirror.oxfordnanoportal.com/software/analysis/ont-guppy_${GUPPYVER}_linux64.tar.gz
tar -xf ont-guppy_${GUPPYVER}_linux64.tar.gz
</syntaxhighlight>