low align rate conflicting with bismark align rate

[yinzhuo0322]

Hello, we encountered some problems when using methylpy to align the data of SRR6911777. The result showed that we only had an align rate which is less than 1%, while we obtained an align rate about 35% by aligning with bismark. Results of other samples were consistent with SRR6911777. This is really confusing and we hope you can give us some suggestions. Our code for methylpy is as follows：
methylpy paired-end-pipeline \ --read1-files $Read1 \ --read2-files $Read2 \ --sample $sampleid \ --forward-ref ${ref_methylpy_mm10}/mm10.genome_f \ --reverse-ref ${ref_methylpy_mm10}/mm10.genome_r \ --ref-fasta ${ref_methylpy_mm10}/mm10.genome.fa \ --path-to-output ${outdir}/methypy_${species} \ --num-procs $threads \ --path-to-picard $picard \ --path-to-samtools $samtools \ --path-to-cutadapt $cutadapt \ --remove-chr-prefix False \ --keep-temp-files True \ --trim-reads True

[yupenghe]

What was the command you used to run bismark? Can you take a quick look at the base composition of read1 and read2 fastq (you can use fastqc too)? Do the reads in read1 fastq have mostly A, T and G? Knowing these would be helpful for troubleshooting.

[yinzhuo0322]

Thanks a lot for your quick reply! Here is my Bismarck code:
bismark --multicore $cores \ --fastq --non_directional --unmapped \ --nucleotide_coverage \ --path_to_bowtie $bowtie2 --bowtie2 \ --genome_folder $ref_bis \ --output_dir $outdir/bismark_${species} \ --temp_dir $outdir/bismark_${species}/temp_bismark_${sampleid} \ -1 $trim_Read1 -2 $trim_Read2
Here are the fastqc data for read1 and read2, it seems that read2 fastq have mostly A, T and G:
read1 fasqc:

read2 fasqc:

[yupenghe]

In you case, this data has the opposite pattern that bismark and methylpy expect from the data from the directional library. For methylpy we will need to use the --pbat option. Similarly bismark also has the --pbat option to handle this data. You were using --non_directional which can work but it is not ideal.

[yinzhuo0322]

Thanks for your sophisticated suggestions! After adding the --pbat option, the align rates are much more normal. We are appreciated very much about your wonderful pipeline and advice. May everything go well with you!