2.6. Methylation profiling analysis
The long-reads and transcriptome mapping results were indexed with
Samtools 1.7.2
(https://github.com/samtools/samtools).
Nanopolish eventalign was designed to work on FAST5 files, which rely on
the HDF5 library, hindering efficient parallel analysis. To address
this, FAST5 files from the previous step were converted to BLOW5 using
slow5tools v.1.0.0 F5c v.1.1 supports BLOW5 and enables the use of
Nanopolish modules for indexing and eventalign. The m6A identification
was performed with m6anet v.2.0.1 , with the -num_iterations 1000 flag.
Results selected for further analysis - comprising the probability of
modification at each position for each transcript - were thresholded at
0.6.