I'm prepping some NGS Illumina data for downstream analysis. To begin, I want to remove any sequencing/ligating adapters and multiplexing (barcoding) tags. To do this, I am using fastx_clipper, which is part of the FASTX-Toolkit. I've also using Trimmomatic for this in the past.
Example command usage: fastx_clipper -Q33 -a TGGAATTCTCGGGTGCCAAGGAACTCCA-mid_tag_insert-AATCTCGTATGCCGTCTTCTGCTTG -l 14 -M 7 -i Input.fastq -o Output.fastq -v -c
Here is my question... Both of these software packages only scan for a single orientation of the adapter you provide within the Illumina reads. However, I find many sequences in all orientations of the adapter, namely: forward, reverse, forward complement, reverse complement. In the forward orientation, the software detects and trims the adapter in >90% of the reads, but in the other 3 orientations the software only detects are trims adapters in ~5% of the reads.
So, is it possible for the adapters to be found in different orientations than the forward sense, or am I seeing artifacts of non-strict adapter matching? Do people usually trim adapters in every possible orientation? Any other suggestions for successfully handling adapters?
Thanks!
