[kevluv93]

@mastal

Thank you for the quick reply! Unfortunately, it's still displaying the same error message. But at least I know the problem is around there, so that's good news.

So, could there be a way to possibly direct trimmomatic to the file without beginning with C:? Or could this possibly be another issue?

I've tried other symbols like brackets and parenthesis just in case 'C:' wasn't it. Same message.

[usad]

Hi

sorry for the inconvenience. At the moment it might be best to not have to use C: at all as trimmomatic looks for data after :
E.g. having the clip file in the actual directory - yukk.

Best Wishes
Björn

[kevluv93]

I figured it would come to that. I think my best option would be to use Linux and not have to direct trimmomatic using "C:". Don't file paths in Linux all begin with a "/home/..."? I'll try that and see if it works.

[Brian Bushnell]

I figured it would come to that. I think my best option would be to use Linux and not have to direct trimmomatic using "C:". Don't file paths in Linux all begin with a "/home/..."? I'll try that and see if it works.

No, not really. The path depends on the system... Java works fine in Windows, and as long as the classpath is set correctly, it should work fine anywhere. You just need to set the "-cp" variable correctly.

[tsangkl]

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

java -jar trimmomatic-0.32.jar PE -threads 11 -trimlog trim_keepbothread.log Hiseq_1.fastq Hiseq_2.fastq Hiseq_1_keeppaired.fastq Hiseq_1_keepunpaired.fastq Hiseq_2_keeppaired.fastq Hiseq_2_keepunpaired.fastq ILLUMINACLIP:NexteraPE-PE.fa:2:30:10:8:TRUE LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36

And my adapter fasta file contain the transposase sequence and primer sequence provided by illumina:

>Trans1_rc
CTGTCTCTTATACACATCTGACGCTGCCGACGA
>Trans2_rc
CTGTCTCTTATACACATCTCCGAGCCCACGAGAC

>Primerprefixi5_rc
GACGCTGCCGACGA
>Primerprefixi7_rc
CCGAGCCCACGAGAC


Thanks

[kevluv93]

Could you guys give me an example script of setting up a classpath for "ILLUMINACLIP:" to get access to the adapter files in the trimmomatic binary download? I should probably mention that my experience with scripting languages begins and ends with "making stick figures in JavaScript", so forgive me if I'm asking for baby steps. I'm still using Windows 8 for this trimmomatic job (it's the OS that all the computers in our college use, so I don't really have a choice.)

@ Bjorn, you mentioned "having the clip file in the actual directory", and that trimmomatic looks for documents after ":". With this info, I deleted the "C:" that came after ILLUMINACLIP: but I got an error message telling me, "ArrayIndexOutOfBoundsException: 1".

@Brian Bushnell, you mentioned that trimmomatic will work on windows as long as I specify the classpaths to the files correctly.

So, could someone give me an example of what I need to write after "ILLUMINACLIP:" to let "ILLUMINACLIP:" get the adapter sequence fa. files that are saved on my desktop? I'm just right clicking these icons and copy/pasting their locations into the trimmomatic script. How else does one make a path to a file?

Thanks for the help! Any input is appreciated!

[Brian Bushnell]

kevluv,

You are not doing anything wrong with Java; your command would fail in any environment. The classpath appears to be correct, you just have the wrong command line.

[MikhailFokin]

Hi guys, seems that Trimmomatic is very useful and performs better that others.

The only thing I can not find, if present at all. Where can I get the info if any adaptor clipping appeared? Yes I can analyse the result by fastqc or go deep to log (still no info for clipping), but is there any obvious place to find adaptor clipping results?

The second question is - what is the clipping/trimming strategy applied for internal (junction Nextera adaptors)?

[MikhailFokin]

could you please give a real example of using -baseout flag?
when using like this "... -phred33 PE S1R1.fatsq S1R2.fastq -baseout outS1.fasta ILLUMINACLIP..." it is still trying to find the list on names afterwards...

[kevluv93]

Haha! I finally got it to work! It was just some user error on my part.

Changes:
Don't use "C:" at the beginning of files.
It works fine with Windows 8
I erased the ".fq" at the end of my input files, I thought I was supposed to specify what file type my input data was, but don't. The name of the file's good enough.

Worked like a charm, it was just user error on my part. Thanks for the help!

[usad]

great that it works now.

[BFM]

Hi i have used trimmomtaic to clip the adapter sequences. Yet i am still having a problem with kmers, sequence per base content. My question is how do we improve the quality if there is any failure in Fastqc results????

[kevluv93]

Hi, back again. When I use trimmomatic I get abnormally high Kmer reads on FastQC, I read out the Kmers and realized that most of my forward adapter was still inside of my cDNA.

I opened the TruSeq2 adapter file and realized that the Prefix PE/1 adapter (I guess that means the forward adapter?) Didn't match the forward adapter I was using, which is:
TruSeq Adapter, Index 2
5’ GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGATGTATCTCGTATGCCGTCTTCTGCTTG

So I went in and replaced the default forward adapter with the sequence you see above.

Now I have another issue, for some reason trimmomatic has managed to cut 8GB of data into 2GB of data. (if I combine the forward and reverse paired and unpaired files) Fastqc is giving me Kmers that are similar to my sequence, and when I opened the forward paired file I saw that fairly large chunks of my primers are still at the 3' end of my cDNA.

ex.
GATCGGAAGAGCACACGTCTGAACTCCAGTCAC
GATCGGAAGAGCACACGTCTGAACTCC
ATCGGAAGAGCACACGTCTGAACTCCAGTCACCGA

They're just small enough for trimmomatic to miss. Is there any helpful suggestions you can give me regarding the settings of trimmomatic to help me cut these small bits of adapters off the end of my reads? Is there a reason why only small fragments of my adapter sequences would be left after using trimmomatic? Finally, is it usual for such a large portion of data to get cut when using trimmomatic or am I screwing this up? (8GB to 2GB of data)

[tonybolger]

Hi, back again. When I use trimmomatic I get abnormally high Kmer reads on FastQC, I read out the Kmers and realized that most of my forward adapter was still inside of my cDNA.

I opened the TruSeq2 adapter file and realized that the Prefix PE/1 adapter (I guess that means the forward adapter?) Didn't match the forward adapter I was using, which is:
TruSeq Adapter, Index 2
5’ GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGATGTATCTCGTATGCCGTCTTCTGCTTG

This sequence looks like a TruSeq-3 adapter - if so, either the TruSeq3-PE or TruSeq3-PE-2 adapter files should remove the sequencing adapters. The useful part of the reads should survive if they the adapters are in the normal position.

If you do have any surviving adapters, can you post a few examples?

Thanks,

Tony.

[Mchicken]

Hey guys,
i`ve got some problems with Trimmomatic:

I have the following 100bp long read:

@HWI-ST365:34625ECACXX:5:1101:3183:2046 1:N:0:TGACCA
NCAGGGGGAACAGGCTGATCTCCCCCAAGAGTCCACATCGACGGGGAGGTTTGGCACCTCGATGTCGGCTCATCGCAACCTGGGGCGGAAGGACGTCCCC
+
#11A?DDDHHBBF?FHBGCGHGGDD@FFDDD9B@FHD8)<FHIG8B89>(,5,5(::<CB?'9@BC8;5?##############################

I only run SLIDINGWINDOW:4:15 on this read

and what i get is:

Log-File:

HWI-ST365:34625ECACXX:5:1101:3183:2046 1:N:0:TGACCA 52 0 52 48

trimmed fastq:

@HWI-ST365:34625ECACXX:5:1101:3183:2046 1:N:0:TGACCA
NCAGGGGGAACAGGCTGATCTCCCCCAAGAGTCCACATCGACGGGGAGGTTT
+
#11A?DDDHHBBF?FHBGCGHGGDD@FFDDD9B@FHD8)<FHIG8B89>(,5


But when i run my own script on the read i can see that the pattern (,5, beginning at position 50 has an average quality of 12.25, which is below the required 15. So the read should survive from position 1 to 49 and not until position 52 as determined by Trimmomatic.

So can anyone tell me where i am wrong?

Thanks
Mchicken