trimming RNA-seq reads with adapter sequence?index? using trimmomatic

Question

[woongjaej]

Hi, guys.

I have a question using trimmomatic to trim adapters from the RNA-seq reads.

I have to QC the reads using trimmomatic but I wonder how it's done.

For example, the sequencing company gave me that the data they gave me were made by "TruSeq Stranded mRNA LT Sample Prep Kit" and say they used "ATTCCT" index.

If I have such info, how do I have to use trimmomatic with these informations?

It would be very helpful if someone can give some advise or pointers.

Thank you very much!.

Woongjae

Answer 1

[Jake Warner]

Hello,
I would recommend first running fastqc to first and check the over-represented sequences. The adapter contamination (and any other artifacts) should show up there. Then you can run trimmomatic tweaking the settings below:

java -jar trimmomatic-0.35.jar PE -phred33 input_forward.fq.gz input_reverse.fq.gz output_forward_paired.fq.gz
output_forward_unpaired.fq.gz output_reverse_paired.fq.gz output_reverse_unpaired.fq.gz ILLUMINACLIP:TruSeq3-PE.fa:2:30:10
LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36

Trimmomatic comes with several pre-built adapter sequences. The ILLUMINACLIP:TruSeq3-PE.fa argument indicates the adapter file to use. You can use TruSeq3-SE.fa for single end data or older adapter files. Additionally, any contaminant sequences you identify from fastqc can be added to a custom trimming file by making an adapters.fa file in the /adapters directory and adding sequences to it in fasta format. Then you pass that to trimmomatic.

More to be learned here.
Good luck!

[woongjaej]

Thanks for your reply Jacob.

I'm wondering if you could give me some advise making custom .fa file with my index info, please.

Is it possible?

Thanks

Woongjae

[Jake Warner]

I'm pretty sure the standard TruSeq3 adapter file will work, but to be sure you can look up your adapters here (page 18) and add them to a fasta file. I would include the universal and index adapter.

[woongjaej]

Thanks a lot!!

I'll try my customized adapter fasta and TruSeq3 file as you suggested, both and then compare them!

Thank you for your kind explainations and pointers Jacob :)

Woongjae