[GenoMax]

Yes. My data is from Illumina MiSeq and my command's line is as follows:
#java -jar /usr/local/bin/trimmomatic-0.30.jar PE -threads 4 -phred33 ./jatoba/Hst_S2_L001_R1_001.fastq ./jatoba/Hst_S2_L001_R2_001.fastq ./jatoba/fq/Hst_S2_PE_1p.fq ./jatoba/fq/Hst_S2_SR_1p.fq ./jatoba/fq/Hst_S2_PE_2p.fq ./jatoba/fq/Hst_S2_SR_2p.fq LEADING:30 TRAILING:30 SLIDINGWINDOW:4:30 HEADCROP:18 MINLEN:20

Thanks.

How about just running:

#java -jar /usr/local/bin/trimmomatic-0.30.jar PE -threads 4 -phred33 -basein jatoba/Hst_S2_L001_R1_001.fastq -baseout ./jatoba/Hst_S2_trim LEADING:30 TRAILING:30 SLIDINGWINDOW:4:30 HEADCROP:18 MINLEN:20

BTW: Are there 3 pairs of samples or are you just providing names of files for holding the output?

[Lays Cruz]

How about just running:

#java -jar /usr/local/bin/trimmomatic-0.30.jar PE -threads 4 -phred33 -basein jatoba/Hst_S2_L001_R1_001.fastq -baseout ./jatoba/Hst_S2_trim LEADING:30 TRAILING:30 SLIDINGWINDOW:4:30 HEADCROP:18 MINLEN:20

BTW: Are there 3 pairs of samples or are you just providing names of files for holding the output?

No. It comes from a only sample. The other files are the outputs, two PE files with the paired sequences and two SR files with single reads removed from R1 and R2 files.

Much appreciate your suggestion, I will try now.

Thanks.

[GenoMax]

Hi all,

I'm testing Trimmomatic's performance on adapter removal, and the results are mysteriously bad. So I'd like to make sure I'm not doing anything wrong. This is my command line (modified from the website):

java -Xmx8g -jar trimmomatic-0.32.jar SE -phred33 dirty.fq tclean.fq ILLUMINACLIP:gruseq.fa:2:30:10

...where dirty.fq is a file containing reads with adapter sequences and gruseq.fa is a file containing the adapter sequences. The adapters are inserted synthetically and the reads are tagged, so I know precisely what the correct results should be, and what I'm getting is not really close. Any suggestions?

The command looks ok, except that you are not providing min adapater length (which defaults to 8). In what way is the output "mysteriously bad"?

[Brian Bushnell]

The command looks ok, except that you are not providing min adapater length (which defaults to 8). In what way is the output "mysteriously bad"?

Most of the adapters didn't get removed.

[Corydoras]

Hi,

Sorry to hijack this post! I got paired-end 150bp RAD sequencing data that I am currently cleaning with Trimmomatic. I just have two quick questions to make sure I am not going wrong anywhere.

1) By the looks of it, most of my adapter contamination occurs within the read. I.e. I have 'P1 -sequence-P1-sequence'. In this case, the single alignment adapter mode will trim this sequence up until the start of the second P1 adapter occurring in the read, leaving me with just 'P1-sequence', am I correct? I am just thinking that I would actually prefer for Trimmomatic to discard these reads entirely, as the reverse part of this read will in all likelihood not come from the same locus as the surviving bit of the forward read? Is there an option to do this? The palindrom mode does not appear to pick these within-read sequences up.

2) Just as a very general question, I also appear to have quite a bit of reverse-complement adapter contamination in my data set and I was wondering if anybody has experience with this for RAD data? Is this something I need to worry about?

Many thanks in advance for any feedback

Sarah

[patouch74]

Hi,

I've chosen trimmomatic for my studies about reads processing.
However I have to justify why I choose this tool instead of others pre processing tools.

Do you have any articles which compare trimmomatic to other tools (except trimmomatic's authors article) from what I can get some informations ?

thanks

[Brian Bushnell]

I have a comparison of adapter trimming here:

http://seqanswers.com/forums/showthread.php?t=42776

...though it's not published or peer-reviewed. I'll be doing another comparison of quality-trimming soon.

Also, here's a paper comparing quality-trimming methods:

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0085024

[Corydoras]

Just in case anybody ever has a similar problem or is confused and stumbles across my post:

Looking closer at my files and where the reverse adapter contamination occurred, it became obvious that the rc sequences were actually simply adapter read through and everything that followed was nonesense which Trimmomatic then perfectly removed. This means in roughly 3% of cases, my fragments were too short for the 150bp HiSeq and the RAD size selection did not work perfectly, but considering it is only 3% and it was my first set of libraries I am fairly happy with that.

Above I stated that I was concerned the forward read would not match the reverse read. Now I believe this is only the case in 100-1000 fragments that consist of tiny fragments with adapter ligating to other tiny fragments of adapter. The majority of the contamination however presents itself in reverse complementary form.

This all obviously rests upon the understanding that when adapter read-through occurs, it will be reverse complementary of the P2 adapters in the forward reads, and reverse complementary of the P1 adapters in the reverse reads. Please feel free to point out if there is something wrong with my logic!

[tsangkl]

Hi, I found trimmomatic very useful.
And it works well with my Hiseq data using Nextera PE adaptor in single end mode.
But I found the output is quite strange in paired end mode:

My output after trimming:
Input Read Pairs: 12484647 Both Surviving: 4943420 (39.60%) Forward Only Surviving: 7297375 (58.45%) Reverse Only Surviving: 16245 (0.13%) Dropped: 227607 (1.82%)

It seems that the forward and reverse reads after trimming is very unbalanced.
What would cause this?
Thanks.

[mastal]

In trimmomatic's default mode, when it trims adapters from paired end reads, it drops the second read of the pair, because the two reads are reverse complements of each other, so the second read doesn't add any extra information.

In newer versions of trimmomatic this can be turned off, so that it keeps both reads after trimming adapters from paired reads. You need to specify 'TRUE' for the <keepBothReads> parameter of the ILLUMINACLIP command.

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

[tsangkl]

Hi, I have successfully trim the transposase sequence from the Hiseq data using trimmomatic, but I would like to trim the primer as well, it only has 15 base long, and I see it is fail to trim it using trimmomatic, do I need to change the seed length of 16 base? How can it be done? Thanks

[mastal]

What was the trimmomatic command that you used, and what sequences did you use in the adapters fasta file?

[kevluv93]

Hi! I think I'm putting in the trimmomatic code incorrectly. I'm using the binary file download of trimmomatic, I'm running these programs off of windows 8 (that might be a problem), and I'm using fastq files that need paired end adapter sequence trimming.

Here is the script I'm using:

java -jar C:\Users\kevluv93\Desktop\Trimmomatic-0.32\Trimmomatic-0.32\trimmomatic-0.32.jar PE -phred33 C:\Users\kevluv93\Desktop\L11of2_S1_L001_R1_001.fastq.fq C:\Users\kevluv93\Desktop\L11of2_S1_L001_R2_001.fastq.fq C:\Users\kevluv93\Desktop\output_forward_paired.fq C:\Users\kevluv93\Desktop\output_forward_unpaired.fq C:\Users\kevluv93\Desktop\output_reverse_paired.fq C:\Users\kevluv93\Desktop\output_reverse_unpaired.fq ILLUMINACLIP:C:\Users\kevluv93\Desktop\Trimmomatic-0.32\Trimmomatic-0.32\TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36

To make things simple, all the files are on my desktop, so if there is an error in the paths I gave trimmomatic to get the files, keep that in mind.

Here is the error message:

Multiple cores found: Using 4 threads
Trimmomatic PE: Started with arguments: -phred33 C:\Users...
Multiple cores found: Using 4 threads
Exception in thread "main" java.lang.NumberFormatException: For input string: "\Users\kevluv93\Desktop\Trimmomatic-0.32\Trimmomatic-0.32\TruSeq3-PE.fa
at java.lang.NumberFormatException.for InputString (unknown source)
at java.lang.Integer.parseInt(Unknown Source)
at org.usadellab.trimmomatic.trim.IlluminaClippingTrimmer.makeIlluminaClippingTrimmer(IlluminaClippingTrimmer.java:53)
at org.usadellab.trimmomatic.trim.TrimmerFactory.makeTrimmer (TrimmerFactory.java:27)

I don't know what is wrong with the script I put in. I used the example on the trimmomatic website as the base for this one, so I thought it would work. Did I input the script correctly? Do the parameters make sense?

New to this, undergrad, greatly appreciative of any input!

[mastal]

@kevluv93

I have not used trimmomatic on Windows, but it looks as if it's expecting what comes after 'C:' to be the second parameter for the ILLUMINACLIP command.

Try putting quotes around the path to the fasta file and see if that helps.

ILLUMINACLIP:'C:\Users\kevluv93\Desktop\Trimmomatic-0.32\Trimmomatic-0.32\TruSeq3-PE.fa':2:30:10

[Brian Bushnell]

You probably have the parameters in the wrong order. Java is parsing "\Users\kevluv93\Desktop\Trimmomatic-0.32\Trimmomatic-0.32\TruSeq3-PE.fa" and expecting an integer, which it is not. I'm not a Trimmomatic expert and I don't know the correct command line, but Trimmomatic should work fine on Windows with the correct command.

If you continue to have problems, send me a pm.