Hi all! I have problem with Trimmomatic 0.33
I have paired-end Illumina reads. For experimenting, I used just 1 read pair, but it comes from a real dataset. So this is my R1.fastq file:
@NS500448:3:H058VAFXX:1:11101:9071:4665 1:N:0:CGTACTAG+ATAGAGAG TCTATCAACAGAGAAAGTTACGCAAAGAAAATGAGTCAAAAGTCTCAAAAAAAGAAGAGTGCTAGGACTGTCTCTTATACACACCGAGCCCACGAGACCGT + AAAAAFFFFFFFFFFFFFFFFFFFFFFFFFFFFAFFFAFFFFFFFFFFFFFFFFFFFFFFFAFFFFFF7FFFFFF<FFFFFF<FFF<FF.FFFFFFFFFFF
And my R2.fastq file:
@NS500448:3:H058VAFXX:1:11101:9071:4665 2:N:0:CGTACTAG+ATAGAGAG TCCTAGCGCTCTTCTTTTTTTGAGACTTTTGACTCATTTTCTTTGCGTAACTTTCTCTGTTGATAGACTGTCTCTTATACGCATATGACGCTGCCGACG + A.<<.F....A).A..<AAF.F)F.<...<F.......AA...<F7FA<..<.A)..FFF.F.<.F.<.F<<.<<<AA7..7....F.7F<7F<.F..F
This is a pair of reads with a nice overlap, both have a piece of adapter on the 3' end. It can be seen from the alignment of R1 and the reverse complement of R2:
EMBOSS_001 1 --------------------------------TCTATCAACAGAGAAAGT 18 |||||||||||||||||| EMBOSS_001 1 CGTCGGCAGCGTCATATGCGTATAAGAGACAGTCTATCAACAGAGAAAGT 50 EMBOSS_001 19 TACGCAAAGAAAATGAGTCAAAAGTCTCAAAAAAAGAAGAGTGCTAGGAC 68 |||||||||||||||||||||||||||||||||||||||||.||||||| EMBOSS_001 51 TACGCAAAGAAAATGAGTCAAAAGTCTCAAAAAAAGAAGAGCGCTAGGA- 99 EMBOSS_001 69 TGTCTCTTATACACACCGAGCCCACGAGACCGT 101 EMBOSS_001 100 --------------------------------- 99
This is my file with adapters adapters.fa:
>Prefix_N702+E502/1 CTGTCTCTTATACACATCTCCGAGCCCACGAGACCGTACTAGATCTCGTATGCCGTCTTCTGCTTG >Prefix_N702+E502/2 CTGTCTCTTATACACATCTGACGCTGCCGACGAATAGAGAGGTGTAGATCTCGGTGGTCGCCGTATCATT
Now when I run Trimmomtaic:
java -jar ~/Trimmomatic-0.33/trimmomatic-0.33.jar PE -phred33 R1.fastq R2.fastq out/R1_pair.fastq out/R1_single.fastq out/R2_pair.fastq out/R2_single.fastq ILLUMINACLIP:adapters.fa:2:30:15
it fails to clip the adapters off. Reads end up unchanged in R1_pair.fastq and R2_pair.fastq
I tried to change pretty much anything I could - threshold values, what is /1 and /2 in adapters.fa (I even tried reverse complements here) and the result is always the same. Interestingly, the only thing that had any effect was to use -phred64 instead of phred33, though I'm sure these files are actually phred 33. When I do so, it trims R1 correctly and discards R2. Though this is the best result I achieved so far, it is of no use, because I want to keep both reads from such pairs and just trim them, and, especially, because my files actually really are phred 33, so when I add any quality-based trimming, Trimmomatic just discards all the reads.
So...does anyone know what am I doing wrong? Thanks.