Editing Nanopore RNA Sequencing Protocol (section)

==Basecalling (Guppy)==

Navigate to the folder with guppy using the <code>cd</code> command (for example, mine is at <code>~/Research/ont-guppy-6.4.2/bin</code>, so I can navigate there using <code>cd ~/Research/ont-guppy-6.4.2/bin</code>, or substituting the path with <code>$GUPPY</code>) and execute the following. Note that the last line of code includes a path to store the log of the ''Guppy'' run, which will require the <code>$GLOGS</code> folder to already have been created.

{{warning|The line <code>-x auto \</code> is only valid for newer versions of ''Guppy'' that supports CUDA GPUs. If you are basecalling with an old version of ''Guppy'', like 3.1.5, or you are on a machine that does not have a CUDA GPU (like a ''Mac''), you’ll want to execute this without this line. You should also remove the line <code>--disable_qscore_filtering</code> with older versions, as this was a newer ''Guppy'' feature.}}

<code>Bash</code>
<syntaxhighlight lang="Bash">
$GUPPY/guppy_basecaller \
--input_path $NRAW/${SAMPLEID} \
--save_path $NBASE/${SAMPLEID} \
--flowcell FLO-MIN106 \
--kit SQK-RNA002 \
--disable_qscore_filtering \
-x auto \
> $GLOGS/${SAMPLEID}_guppy${GUPPYVER}_$(date +"%Y%m%d%H%M%S").txt
</syntaxhighlight>

Note that the lines containing <code>--flowcell</code> and <code>--kit</code> can be replaced with a configuration (e.g. <code>--config rna_r9.4.1_70bps_hac.cfg</code>). <code>-x auto</code> specifies that you want to run the basecaller using the default GPU (remove this line if you want to do CPU, or replace <code>auto</code> with <code>cuda:X</code>, where <code>X</code> is the GPU identifier you want to use). The last line just exports the output as a text file for logging in case you ever need to reference it later, with the ID of your sample and the current date and time stamped in by the system. You can exclude this if you wish.