TRIMMOMATRIC Nextera trimming

Question

[meekadi]

Hi, can anyone help? I started using trimmomatic 0.33 today so I'm not that familiar with it. I have paired end data and after checking the quality of my reads I tried trimming for the adapters and low quality using trimmomatic. the adapters used for the my sequences are nextera. I followed the example that was used online for trimming Truseq adapters on an ngs and after trimming the adapters were still detected in my sequences. this is the command I used for my run:

java \
  -phred trimmomatic-0.33.jar PE \
  -phred 33 \
  TW_R1.fastq.gz TW_R2.fastq.gz \
  TW_R1trimmed.fastq.gz TW_R2trimmed.fastq.gz \
  ILLUMINACLIP:NexteraPE-PE.fa:2:30:10: \
  LEADING: 3 \
  SLIDINGWINDOW:4:15 \
  MINLEN:40

[Biomonika (Noolean)]

Could this help: http://sites.psu.edu/biomonika/2015/07/03/trim-now-or-never-pe-and-mp-adaptor-removal/ (disclaimer: points to my own blog)

[meekadi]

Thanks Noolean. I read the script. Will try and see which one works for me

[Ashutosh Pandey]

You also need to specify output files for orphan reads (read from a read-pair where the other read got removed). Otherwise the command looks good to me. You should compare the quality profile of fastq files before and after trimming. After you are done with trimming, you can use FastQC to make sure if the adaptors have been removed completely otherwise they will show as over represented sequences in your fastq files. You can also check the quality of reads etc.

[meekadi]

Thank you. I trimmed my data again adding what you recommended. I then compared my sequences before and after trimming using fastqc and there is still no adapters in the trimmed reads but the content decreased. I'm wondering if it has anything to do with the base position.