Can't get paired end data to align with Bowtie

[Misa]

Hi to all of you,
I'm more or less brand new at bioinformatics, eager to learn, but tend to end up mostly feeling stupid.

I was given a set of miRNA paired end files (run on Illumina NextSeq 500) to identify novel miRNAs as potential biomarkers in blood. Before I started to think too much of HOW to actually do the identification and differential analysis of those sequences I read up on alignment tools. So I ended up aligning my files with Bowtie2 after trimming with cutadapt. Got an overall alignment rate of around 94% for all files.

Then I came across miRDeep2 and saw the potential with that pipeline. Since I also want to learn I tried simply to align my files with Bowtie first and thought I could convert them before feeding them into the pipeline. However, for some reason I get only 1% alignment rate for my reads. Same files I got 94% alignment rate for with Bowtie2. I tried to align just one of the paired end files and got an alignment rate of over 80%. So why can't I get the paired end files to align as nicely? Any suggestions? Now that I DO have paired end it feels like a waste to only use one of the files as "single end".

Bowtie for paired end:

./bowtie -n 0 -l 15 -e 80 RefCat -q -1 /pathtomystorage/trimmed/trimmed_1.fq -2 /pathtomystorage/trimmed/trimmed_2.fq -S /pathtomystorage/alignment/bowtie_alignment.sam

Result:
# reads processed: 10229619
# reads with at least one reported alignment: 91017 (0.89%)
# reads that failed to align: 10138602 (99.11%)
Reported 91017 paired-end alignments to 1 output stream(s)


Bowtie for "single end":

./bowtie -a --best -n 0 -l 15 -e 80 RefCat -q /pathtomystorage/trimmed/trimmed_1.fq -S /pathtomystorage/alignment/bowtie_alignment_1_.sam

Result:
# reads processed: 10229619
# reads with at least one reported alignment: 8242967 (80.58%)
# reads that failed to align: 1986652 (19.42%)
Reported 73843253 alignments to 1 output stream(s)

I have tried to adjust the parameters for -X, -I and --ff/--rf/--fr without much improvement...

[GenoMax]

Have you scanned trimmed these data files. Since you are working with miRNA they are likely to have illumina adapters in reads. Since bowtie does not do gapped alignment it is likely not able to align the reads.

Your reads probably will overlap (since the inset size must be small). You could merge the R1/R2 reads (using a tool like bbmerge) before you do scan/trimming followed by alignments.

[Misa]

Have you scanned trimmed these data files. Since you are working with miRNA they are likely to have illumina adapters in reads. Since bowtie does not do gapped alignment it is likely not able to align the reads.

Your reads probably will overlap (since the inset size must be small). You could merge the R1/R2 reads (using a tool like bbmerge) before you do scan/trimming followed by alignments.

Thank you for your suggestion. I had trimmed the reads for adapters prior to alignment and find it strange I can align 94% with Bowtie2 but only 1% with Bowtie. Would you suggest I merge the trimmed files, or should I merge the untrimmed files?

Thnx!

[Misa]

Well, merging reads prior to trimming with default options for BBMerge only merged 3.2% of my reads. 96.7% ended up as ambiguous, at least for one of the samples.

[Brian Bushnell]

For BBMerge with micro-RNA, you need to add the flag mininsert=17. The default is 35, which is too long for micro-RNA libraries.

[Misa]

For BBMerge with micro-RNA, you need to add the flag mininsert=17. The default is 35, which is too long for micro-RNA libraries.

Thank you kindly for that suggestion, I now joined 94.2% of the reads instead of 3%