Hey all,

I'm just getting into rna-seq analysis and have run into a problem that's is very confusing. I ran multiplexed RNAseq samples on a Hiseq, paired end, 100bp, reads. The data was demultiplexed and what I thought was trimmed (I used illumina truseq index primers).

So I run my R1 & R2 reads on fastqc and find ~20% illumina universal adapter sequences at the 3' end of my reads. I run BBDuk and it removes about 10% so I'm left with ~ 10% still contaminating the reads. Im wondering what I should do next???

The initial adapter content was a linear slope starting from position 55 to 85 bp, after BBduk the value went to 10% sloping up from 72 to 85bp. Links to adapters image graph


I'm not sure how to get rid of the rest of the adapters. Fastqc has also changed from an X to an ! So that might be ok then?

$ ./bbduk.sh in1=S42ND_L002_R1_001.fastq.gz in2=S42ND_L002_R2_001.fastq.gz out1=S42ND_L002_R1_001_TRIMMED.fastq.gz out2=S42ND_L002_R2_001_TRIMMED.fastq.gz ref=adapters.fa ktrim=r k=23 mink=11 hdist=1 tpe tbo
java -ea -Xmx1205m -Xms1205m -cp /media/sf_ubuntushared/bbmap/current/ jgi.BBDuk in1=S42ND_L002_R1_001.fastq.gz in2=S42ND_L002_R2_001.fastq.gz out1=S42ND_L002_R1_001_TRIMMED.fastq.gz out2=S42ND_L002_R2_001_TRIMMED.fastq.gz ref=adapters.fa ktrim=r k=23 mink=11 hdist=1 tpe tbo
Executing jgi.BBDuk [in1=S42ND_L002_R1_001.fastq.gz, in2=S42ND_L002_R2_001.fastq.gz, out1=S42ND_L002_R1_001_TRIMMED.fastq.gz, out2=S42ND_L002_R2_001_TRIMMED.fastq.gz, ref=adapters.fa, ktrim=r, k=23, mink=11, hdist=1, tpe, tbo]

Version 38.68

Thanks for any help