Editing Nanopore RNA Sequencing Protocol (section)

==Formatting (Samtools)==

Next we need to convert the exported <code>.sam</code> file to binary (<code>.bam</code>), which is required for some of the steps we’re going to perform down the road for ''m6A detection''. Then, we will need to sort the <code>.bam</code> file (in other words, organize the mapped reads by chromosome and position), and then index it by generating a <code>.bam.bai</code> file.

<code>Bash</code>
<syntaxhighlight lang="Bash">
cd $NALI/${SAMPLEID}
samtools view -bS ${SAMPLEID}_taligned_unsorted.sam > ${SAMPLEID}_taligned_unsorted.bam
samtools sort ${SAMPLEID}_taligned_unsorted.bam -o ${SAMPLEID}_taligned.bam
samtools index ${SAMPLEID}_taligned.bam ${SAMPLEID}_taligned.bam.bai
</syntaxhighlight>

===Isolate Chromosome===

{{note|This step is ''ONLY'' for alignments that have been done to the genome and for processing with ''EpiNano''. If you are using ''m6ANet'', you’ll want to skip this portion.}}

This will speed up the ''EpiNano'' processing step substantially, since it will not be analyzing ''all'' of your data at once. ''Note that we defined the chromosome we wanted to isolate above at the start of the protocol'', with <code>${CHR}</code>.

<code>Bash</code>
<syntaxhighlight lang="Bash">
samtools view -b ${SAMPLEID}_aligned.bam ${CHR} > ${SAMPLEID}_${CHR}.bam
samtools index ${SAMPLEID}_${CHR}.bam ${SAMPLEID}_${CHR}.bam.bai
</syntaxhighlight>

===Check Read Count===

''Samtools'' comes with the ability to check the total number of reads in a <code>.bam</code> file via the command <code>view -c</code>. Therefore, at this stage you should count the total number of reads in both the master aligned file and (if aligned to the genome) the individual chromosome you isolated. This can be done via the following:

<code>Bash</code>
<syntaxhighlight lang="Bash">
samtools view -c ${SAMPLEID}_aligned.bam
samtools view -c ${SAMPLEID}_${CHR}.bam
</syntaxhighlight>

Or, if mapped to the transcriptome:

<code>Bash</code>
<syntaxhighlight lang="Bash">
samtools view -c ${SAMPLEID}_taligned.bam
</syntaxhighlight>

For reference, the <code>FAM95931</code> sample (identified here by ''flow cell'' ID) we ran had about 1.9 million reads total and 106,000 reads across chromosome 19. This was using a standard ''MinION'' flow cell, ''not'' a flongle, which should have about 120-200k reads.