[Ahaswer]

Hi Brian. Yes, the reads have been adapter-trimmed. Moreover alignment toward long sequences (whole reference) gives good alignment rate and low while using short (miRBase) sequences.

[Brian Bushnell]

Ahhh... to clarify, what I meant was NOT adapter trimming reads could cause the bias, but I guess that's out if the reads were successfully trimmed.

[moses122]

I got this problem too. Did you figure out why exactly alignment is better for long sequences than miRBase?

[mziemann]

I got this problem too. Did you figure out why exactly alignment is better for long sequences than miRBase?

Please don't align directly to the mature.fa, only about 50% of miR reads are the exact mature sequence, you are missing a lot of sequence diversity that will map clearly to the harpin.fa. Calling 3p and 5p arm switching on the other hand, that's a different story.

[moses122]

Please don't align directly to the mature.fa, only about 50% of miR reads are the exact mature sequence, you are missing a lot of sequence diversity that will map clearly to the harpin.fa. Calling 3p and 5p arm switching on the other hand, that's a different story.

Thanks for replying. I did mapped to hairpin.fa. The alignment rate is only around 0.01% and it's 90% for genome. I used bowtie and allowed one mismatch.