[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.

[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 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!!!

[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.

[Niloofar]

OK, Thanks Westerman

[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]

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

[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.

[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?

[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

[cllorens]

Hi Tony thank you for trimmomatic.

A question of newie about the manual.


When using the paired End Mode (below is as it is in your example of the manual)

java -classpath <path to trimmomatic jar> org.usadellab.trimmomatic.TrimmomaticPE [-threads <threads>] [-phred33 | -phred64] [-trimlog <logFile>] <input 1> <input 2> <paired output 1> <unpaired output 1> <paired output 2> <unpaired output 2> <step 1> ...


The question is which information is reported in the outputs "unpaired output 1" and "unpaired output 2" I installed the tool and I trying to make a couple of things with it but the files I have in return from the outputs are empty. Sure I am doing something wrong
but i could earn some time if you clarify me this doubt.

Best
Carlos

[tonybolger]

Ok, thanks!!! But now I have another question. So with a score of 15 there has to be 25 bp of adapter sequence for anything to get trimmed.

Yep.

There's a trade-off:- you can set it lower, but then you will naturally be more likely to get something 'adapter-like' by random chance. I wouldn't recommend going below around 10 though.

[ETHANol]

Ok, thanks!!! But now I have another question. So with a score of 15 there has to be 25 bp of adapter sequence for anything to get trimmed.

[tonybolger]

Can someone help me understand the perimeters for ILLUMINACLIP.
I don't understand what the value for 'palindromeClipThreshold' or 'simpleClipThreshold' represent. In the example code they use values of 40 and 15, respectively but I have no idea what these numbers are doing. Also, does the value specified for 'palindromeClipThreshold' do anything in single end mode?

Sorry about the confusion.

These values are (very roughly speaking) log-10 probabilities of getting a match at random. Each perfectly matching base scores just over 0.6, so 15 requires a perfect 25 base match. Each mismatching base reduces the score by the Q/10 value of that base. So it takes 5 or even 6 additional matching bases to overcome one high quality mismatch, but maybe only 1 or 2 additional bases if the mismatching base is low quality.

"Palindrome" mode is only used in paired mode, when appropriately named "prefix" sequences are provided, and since it does 'end to end' matching of the pairs, it can afford a higher threshold, as suggested.

[ETHANol]

Can someone help me understand the perimeters for ILLUMINACLIP.
I don't understand what the value for 'palindromeClipThreshold' or 'simpleClipThreshold' represent. In the example code they use values of 40 and 15, respectively but I have no idea what these numbers are doing. Also, does the value specified for 'palindromeClipThreshold' do anything in single end mode?

Thanks

ILLUMINACLIP:<fastaWithAdaptersEtc>:<seed mismatches>:<palindrome clip threshold>:<simple clip threshold>

fastaWithAdaptersEtc: specifies the path to a fasta file containing all the adapters, PCR sequences etc. The naming of the various sequences within this file determines how they are used. See below.

seedMismatches: specifies the maximum mismatch count which will still allow a full match to be performed

palindromeClipThreshold: specifies how accurate the match between the two 'adapter ligated' reads must be for PE palindrome read alignment.

simpleClipThreshold: specifies how accurate the match between any adapter etc. sequence must be against a read.