Reverse Sequenced Reads

[suzziek]

Hello!

Recently I sequenced some methylation libraries, single end mode, in the reverse direction (from the i7 side). Is it correct to input the genome.fasta file as is and run the pipeline in non-directional mode or Is it correct to input the reverse complement of the genome.fasta file and run the pipeline in directional mode?

Both runs result in similar mapping efficiency but I wonder if the wrong C>T and G>A conversions are happening for the negative strand when the CX report is being calculated? Here is the code I use post trimming and fastqc:

bismark_genome_preparation /path/to/file/ --verbose bismark --non_directional --genome /path/to/file/ *_trimmed_trimmed.fq bismark_methylation_extractor --bedGraph --CX --counts --cytosine_report --genome_folder /path/to/file/ *bt2.bam

Any suggestions would be greatly appreciated. Cheers.