[cllorens]

Hi again,
Do not need answer for my question. I found this post where the author answer a similar question. Thank you anyway.

http://seqanswers.com/forums/showthread.php?t=10003

Carlos

[shawpa]

quality values for trimmomatic

I am having trouble understanding the quality value used in trimmomatic. I want to trim bases from 3' and 5' ends that are below Q20. I have Illumina 1.8+ so my quality values are phred64. For the sample script they use 3 as the quality value. This can't be a Qscore of 3 so what is this?

[shawpa]

I am having trouble understanding the quality value used in trimmomatic. I want to trim bases from 3' and 5' ends that are below Q20. I have Illumina 1.8+ so my quality values are phred64. For the sample script they use 3 as the quality value. This can't be a Qscore of 3 so what is this?

Please disregard. I was being stupid. My values are in phred33.

[Niloofar]

Trimming 3' end with Trimmomatic

Hi,

Would someone please tell me if there is a way to do trimming in Trimmomatic (using sliding window) on 3' end only?

Thanks

[westerman]

Take the example on the web page and only use the 'TRAILING:3 SLIDINGWINDOW:4:15' part of it. I.e., drop the 'ILLUMINACLIP:illuminaClipping.fa:2:40:15 LEADING:3 MINLEN:36' part of the example.

[Niloofar]

OK, Thanks Westerman

[alisrpp]

I have some questions about the folder with the sequences of the adapters:

1. Where i have to create the folder?
2. Can anyone give me an example of the format? I'm using Illumina TruSeq adapters.

Thanks.

[alisrpp]

Hi all,

I started to use Trimmomatic 0.22 three weeks ago and i'm having some difficulties.

Until now i was doing the adapter trimming and quality trimming with CLC. In order to check which method is going to give me the better quality of trimmed reads i'm doing again the trimming of my libraries with Trimmomatic and comparing the fastQC reports of both (CLC and Trimmomatic).

For some reason Trimmomatic is not doing a very good job with my forward sequences and i'm still having some overrepresented sequences that the fastQC recognize as one of the Illumina indexes.

I thought that maybe was because my adapters fasta file was wrong so i was playing around a little bit with it.

Now i'm completely lost and exhausted so i need help. Here i attached my 2 adapters fasta files. Because i built my libraries with 2 different kits from Illumina (the Multiplexing Sample Prep Oligo Only kit: with homemade adapters and with Illumina adapters; and the TruSeq kit).

And here is my script:

for FRW in $(ls *_R1_005.fastq.gz); do
for RVS in $(ls *_R2_005.fastq.gz); do
bsub -q $queue java -Xincgc -Xms4g -Xmx4g -classpath ../../trimmomatic-0.22/Trimmomatic-0.22/trimmomatic-0.22.jar org.usadellab.trimmomatic.TrimmomaticPE -phred33 $FRW $RVS ../trimmomatic_data_trial/$FRW.FRWPE.fq.gz ../trimmomatic_data_trial/$FRW.FRWUnP.fq.gz ../trimmomatic_data_trial/$RVS.RVSPE.fq.gz ../trimmomatic_data_trial/$RVS.RVSUnP.fq.gz ILLUMINACLIP:../../trimmomatic-0.22/TruSeq_adapters.fa:2:40:15 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:20

THANKS!!!

[westerman]

Well, your script is confusing. It looks like you'll be comparing multiple forward files to multiple reverse files without any correspondence between the forward and reverse sequences. You then regenerate the same output files over and over again with the last files being generated good for the reverse sequences but not the forward ones.

Now if you only have one matching file for your 'ls' the above observation doesn't really matter. But if you have multiple files then ... let's see.

Assume the following files:
A_R1_005.fastq.gz
B_R1_005.fastq.gz
C_R2_005.fastq.gz
D_R2_005.fastq.gz

Which I'll abbreviate as 'A', 'B', 'C', and 'D.

A vs C produces files:
A.FRWPE.fq.gz A.FRWUnP.fq.gz C.RVSPE.fq.gz C.RVSUnP.fq.gz

A vs D produces files: (bad match between R1 and R2)
A.FRWPE.fq.gz A.FRWUnP.fq.gz D.RVSPE.fq.gz D.RVSUnP.fq.gz

B vs C produces files: (bad match between R1 and R2)
B.FRWPE.fq.gz B.FRWUnP.fq.gz C.RVSPE.fq.gz C.RVSUnP.fq.gz

B vs D produces files:
B.FRWPE.fq.gz B.FRWUnP.fq.gz D.RVSPE.fq.gz D.RVSUnP.fq.gz

Everything is confused!

Now it is quite possible that your script is working ok (either because of single files or because it was not presented completely) but you may wish to run your Trimmomatic without the loops involved.

[alisrpp]

Hi westerman,

Thanks for the answer.

So, i only have one forward file (R1) and one reverse file (R2) so i think that even is better to not to use loops the script is not the problem. Before using this script with the loops i was using this:

java -Xincgc -Xms4g -Xmx4g -classpath ../../trimmomatic-0.22/Trimmomatic-0.22/trimmomatic-0.22.jar org.usadellab.trimmomatic.TrimmomaticPE -phred33 [name_forward_file] [name_reverse_file] ../trimmomatic_data_trial/[name_forward_file]_PE.fq.gz ../trimmomatic_data_trial/[name_forward_file]_UnP.fq.gz ../trimmomatic_data_trial/[name_reverse_file]_PE.fq.gz ../trimmomatic_data_trial/[name_reverse_file]_UnP.fq.gz ILLUMINACLIP:../../trimmomatic-0.22/TruSeq_adapters.fa:2:40:15 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:20

... and i was having the same problem with my reverse trimmed sequences. That's why i thought that the problem is in my adapters fasta file.

[kmcarr]

Your adapter fasta files have /2 after the IDs of the index adapters which tells Trimmomatic to only check these sequences against the second (reverse) read. These adapters will show up in the first (forward) read, not the second. Likewise the universal adapter sequence will show up in the second read, not the first. You need to swap the /1 and /2 endings in your adapter FASTA files and you should use the reverse complement of the Universal adapter. You could remove /1 and /2 entirely forcing Trimmomatic to check the adapters against all reads but that is inefficient.

[westerman]

Ok, having gotten the possible script problem out of the way, everything else looks syntactically correct. Time to dig into the adapter file or the actual reads that are not being properly trimmed. It is possible that the 2:40:15 parameter to the ILLUMINACLIP is tripping you up. Are you using the simple trim or the, in my experience more rare, palindrome trim? You say:

i'm still having some overrepresented sequences that the fastQC recognize as one of the Illumina indexes.

What are these sequences?

Can you look at a handful of reads reported to have illumina indexes and manually see if they have the index. And, if so, how good is the match?

[westerman]

Your adapter fasta files have /2 after the IDs of the index adapters which tells Trimmomatic to only check these sequences against the second (reverse) read. ...

Ah, good catch! All my adapters are non-strand specific with reverse-complements thus I just skipped over that part of his adapters. It is undoubtedly the correct solution.

[alisrpp]

Thanks a lot!!!

Do you recommend me to do an individual FASTA file for each index?

[westerman]

All my adapters are in one file. Given the number of adapters I can not see it working otherwise. I undoubtedly over process my sequences by looking at both strands in both directions with all possible adapters -- as kmcarr says, "inefficient" -- but it makes me more comfortable that I am picking up everything.