Hi @galaxyNewbie and @Jennifer Hillman Jackson,
I am having the same problem: using galaxy, i selected the option "Perform initial ILLUMINACLIP step" and then instead of using the adapters sequences by default (any of those provided), i provided custom adapter sequences (meaning I added in the box, in multifasta format, all the adapters I wanted to trim, plus PCR primers employed to get the amplicons used to prepare my libraries). After running trimmomatic (with default settings and the algorithms SLIDINGWINDOW and MINLEN), and checking the output data with FastQC (adding a contaminant list with all my PCR primers in multifasta format), I still see a lot of overrepresented sequences which overlap with some of the primers that should have been trimmed away with trimmomatic.
Therefore, my question is: why is trimmomatic not cutting those sequences if they were specified together with the adaptors and other primers which seem to have cut well? How do I get rid of those? For the explanation please consider I am new to Gallaxy and that I have no experience with command lines. Thanks in advance for any help!
Please see below the result of the FasQC of one of such examples after running Trimmomatic:
Sequence Count Percentage Possible Source
ATGGAGACCAAGCTCATCACGTGGGGAGCAGACACCGCCGCGTGCGGTGA 19551 4.748846247267427 NS3-1a-Fext (100% over 21bp)
GATATGCGAGGTGCTGAGCGACTTCAAGACCTGGCTAAAAGCCAAGCTCA 3471 0.8430896283701724 No Hit
TCCTTCCTTGTGTTCTTCTGTTTTGCATGGTATCTGAAGGGTAAGTGGGT 2527 0.613796453728443 No Hit
TCCTTCCTTGTGTTCTTCTGCTTTGCATGGTATCTGAAGGGTAAGTGGGT 2188 0.5314549429196016 No Hit
GATATGCGAGGTGCTGAGCGACTTTAAGACCTGGCTAAAAGCCAAGCTCA 1937 0.4704882195773622 No Hit
GACGTCGTGTGCTGCTCGATGTCTTACTCCTGGACAGGCGCACTCGTCAC 1285 0.31212047607481175 NS5B-1a-fwd (95% over 23bp)
GAGCAGCACACGACATCCTCCGTGCCAGCCTCACTACTAACTGTTGACCA 1273 0.30920573232936605 No Hit
GTAAAACGACGGCCAGTGAATTCTAATACGACTCACTATAGC 1162 0.28224435268399317 No Hit
CATCGCGTACGCCAAGATCATGGTAGCCGTGGGCGACCAGTTCATCATCA 1125 0.27325722613553555 No Hit
GAGCAACACACGACGTCCTCCGTGCCAGCCTCACTACTAACTGTTGACCA 848 0.2059752246781637 No Hit
ATGGAGACCAAGCTCATCACGTGGGGAGCAGACACCGCCGCGTGCGGCGA 761 0.1848433325236823 NS3-1a-Fext (100% over 21bp)
GATGTCGTGTGCTGCTCGATGTCTTACTCCTGGACAGGCGCACTCGTCAC 699 0.16978382317221277 NS5B-1a-fwd (95% over 23bp)
GACGTCGTGTGCTGCTCGATGTCTTATTCCTGGACAGGCGCGCTTATCAC 667 0.16201117318435754 NS5B-1a-fwd (95% over 23bp)
ATGGAGACCAAGCTCATCACGTGGGGAGCAGACACCGCCGCGTGCGGTGATATCATCAACGGCTTG 656 0.1593393247510323 NS3-1a-Fext (100% over 21bp)
ATGGAGACCAAGCTCATCACGTGGGGAGCGGACACCGCCGCGTGCGGTGA 638 0.15496720913286374 NS3-1a-Fext (100% over 21bp)
GAGCAACACACGACATCCTCCGTGCCAGCCTCACTACTAACTGTTGACCA 486 0.11804712169055137 No Hit
GAGCAGCACACGACATCCTCCGCGCTAGCCTCACTACTAACTGTTGACCA 430 0.10444498421180472 No Hit
