[mgbfx9]

Hi Dr. Bolger,

Thanks for the Trimmomatic tool. I want to use the tool and just downloaded Trimmomatic Version 0.33 on my computer. But reading through its content and the manual, I was wondering whether I can use the tool for preprocessing of my Illumina PE MiSeq data? Because in its adapters directory, I see all TruSeq files, but not MiSeq. Could it be inside NexteraPE-PE.fa? However my DNA library was made by Nextera XT system.
If your answer is 'yes', then do I have to change the name in my code from TruSeq3-PE.fa to NexteraPE-PE.fa ?

Thank you in advance for your response!

[kmcarr]

Hi Dr. Bolger,

Thanks for the Trimmomatic tool. I want to use the tool and just downloaded Trimmomatic Version 0.33 on my computer. But reading through its content and the manual, I was wondering whether I can use the tool for preprocessing of my Illumina PE MiSeq data? Because in its adapters directory, I see all TruSeq files, but not MiSeq. Could it be inside NexteraPE-PE.fa? However my DNA library was made by Nextera XT system.
If your answer is 'yes', then do I have to change the name in my code from TruSeq3-PE.fa to NexteraPE-PE.fa ?

Thank you in advance for your response!

mgb,

You are confusing library preparation method with instrument used to actually sequence the library.

Libraries may be prepared using TruSeq or Nextera (or any number of other vendor's compatible kits) and those libraries may then be sequenced on any Illumina instrument (HiSeq, MiSeq, NextSeq). Which instrument was used to generate the data has no bearing at all on processing the reads post run. For trimming you only need to know what kit was used to prepare the libraries.

[mgbfx9]

kmcarr,

Thank you for the response of my post. I am newbie, so I appreciate your explanation.

The DNA libraries of my samples were made by Nextera XT system. The DNA libraries were run paired end in Illumina MiSeq instrument.

The doubt I had, TruSeq libraries are made for RNA seq, so I was wondering whether this program could be run for DNA dataset? However, I didn't see that the program is only for the RNA dataset in the Manual or in the reference paper. So, my question was what should I write in place of

ILLUMINACLIP:TruSeq3-PE.fa

Although I was trying to run and execute the program from the directory where I have Trimmomatic jar according to the Manual for the paired end, but I got following response:

Error: Unable to access jarfile trimmomatic-0.33.jar

I appreciate any help. Thanks,

[mastal]

If your librairies were prepared using the Nextera XT kit, then you need a file with the adapter sequences for Nextera XT, not TruSeq.

try specifying the full path to your trimmomatic.jar file.

[mgbfx9]

Thank you, Mastal for your response to my post.

I have few questions:
1. I have downloaded Trimmomatic from

http://www.usadellab.org/cms/index.php?page=trimmomatic

when I saw the file Trimmomatic-Src-0.33.zip and the index.php?page=trimmomatic in my directory

2. I unzipped the file, Trimmomatic-Src-0.33.zip when I got Trimmomatic 0.33,
3. I wanted to see first whether I can execute Trimmomatic, So, from my directory where I have my Trimmomatic downloaded, I ran the following code:
java -jar trimmomatic-0.33.jar
Error: Unable to access jarfile trimmomatic-0.33.jar
Which I feel normal as my file doesn’t have extension .jar

4. So, I changed, and ran:
/home/mydirectory/java –jar trimmomatic-0.33

Error: Unable to access jarfile trimmomatic-0.33.jar

I think probably my download is not complete. Any suggestions?
Thank you in advance,

[mastal]

If the location of the trimmomatic jar file isn't in your PATH, the computer won't know where to look for it.

If you specify the full path, starting from the root directory, then the computer will know where to look.

If you are trying to run trimmomatic from the directory where your trimmomatic.jar file is, you can also try

java -jar ./trimmomatic-0.33.jar

[mgbfx9]

Thank you, mastal for the suggestion.

I ran from Trimmomatic directory, the code you gave, but still gave me the Error message.

[mydirectory@cluster trimmomatic-0.33]$ java -jar ./trimmomatic-0.33.jar

Error: Unable to access jarfile ./trimmomatic-0.33.jar

[westerman]

When a relative path does not work (e.g., the './') then trying the full path is recommended.

I run as follows:

java -Xms512m -Xmx4000m -classpath [full_path_to_jar] org.usadellab.trimmomatic.Trimmomatic[either PE or SE]

[GenoMax]

Thank you, mastal for the suggestion.

I ran from Trimmomatic directory, the code you gave, but still gave me the Error message.

[mydirectory@cluster trimmomatic-0.33]$ java -jar ./trimmomatic-0.33.jar

Error: Unable to access jarfile ./trimmomatic-0.33.jar

Can you post a listing of the directory you issued the above command from?

$ ls -l

[mgbfx9]

Thank you westerman and GenoMax for your response.

I found out that I downloaded not the Trimmomatic binary file. So, I downloaded now the binary file, and found that it contains trimmomatic-0.33.jar file. So when I ran Trimmomatic from its directory,

[myhomedir Trimmomatic-0.33]$ java -jar trimmomatic-0.33.jar
Usage:
PE [-threads <threads>] [-phred33|-phred64] [-trimlog <trimLogFile>] [-basein <inputBase> | <inputFile1> <inputFile2>] [-baseout <outputBase> | <outputFile1P> <outputFile1U> <outputFile2P> <outputFile2U>] <trimmer1>...
or:
SE [-threads <threads>] [-phred33|-phred64] [-trimlog <trimLogFile>] <inputFile> <outputFile> <trimmer1>...

So, I think it is alright, let’s see, how this run with my PE samples, hopefully it would be okay. Thanks,

[GenoMax]

Perfect. Things should go much better now.

[mgbfx9]

Thank you GenoMax.
I ran Trimmomatic from its directory, and I am not sure whether it ran properly. Because there are lot of comments and at the end, reported “Trimmomatic completed successfully”.

Here is my code:
[mydir Trimmomatic-0.33]$ java -jar trimmomatic-0.33.jar PE -threads 8 -phred33 /home/mydir/ngs_trimm/sample1_L001_R1_001.fastq /home/mydir/ngs_trimm/sample1_L001_R2_001.fastq
/home/mydir/ngs_trimm/output_forwardP.fastq /home/mydir/ngs_trimm/output_forwardU.fastq
/home/mydir/ngs_trimm/output_reverseP.fastq
/home/mydir /ngs_trimm/output_reverseU.fastq ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36

Output:
TrimmomaticPE: Started with arguments: -threads 8 -phred33 /home/mydir/…. started with my full codes
Oct 26, 2015 5:18:10 PM org.usadellab.trimmomatic.trim.IlluminaClippingTrimmer makeIlluminaClippingTrimmer
SEVERE: null
java.io.FileNotFoundException: /home/mydir/Trimmomatic-0.33/TruSeq3-PE.fa (No such file or directory)
at java.io.FileInputStream.open(Native Method)
at java.io.FileInputStream.<init>(FileInputStream.java:146)
at org.usadellab.trimmomatic.fasta.FastaParser.parse(FastaParser.java:54)
at org.usadellab.trimmomatic.trim.IlluminaClippingTrimmer.loadSequences(IlluminaClippingTrimmer.java:107)
at org.usadellab.trimmomatic.trim.IlluminaClippingTrimmer.makeIlluminaClippingTrimmer(IlluminaClippingTrimmer.java:70)
at org.usadellab.trimmomatic.trim.TrimmerFactory.makeTrimmer(TrimmerFactory.java:27)
at org.usadellab.trimmomatic.TrimmomaticPE.run(TrimmomaticPE.java:496)
at org.usadellab.trimmomatic.Trimmomatic.main(Trimmomatic.java:35)

Input Read Pairs: 3032230 Both Surviving: 3018684 (99.55%) Forward Only Surviving: 12217 (0.40%) Reverse Only Surviving: 1236 (0.04%) Dropped: 93 (0.00%)
TrimmomaticPE: Completed successfully

What is the meaning of this line : java.io.FileNotFoundException: /home/mydir/Trimmomatic-0.33/TruSeq3-PE.fa (No such file or directory)

I have seen the output files in my folder, but when I viewed them with the untrimmed, I see some of the bases were missing, and not the N’s or H’s or + sign, I have the doubt, did it really trimmed the places I want to.

I think I am almost there, so any help is really appreciated. Thanks,

[GenoMax]

That adapter file is not in the same directory as the jar file so you should change the last part of your command to include the location of the adapter file

ILLUMINACLIP:/home/mydir/Trimmomatic-0.33/adapters/TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36

[mastal]

@mgbfx9
If you want to know whether trimmomatic is trimming your reads in the way you intend it to,
it is helpful to get trimmomatic to produce a logfile, which specifies how each read has been trimmed.

See the trimmomatic webpage,

http://www.usadellab.org/cms/index.php?page=trimmomatic

and add the option '-trimlog logfile_name' to the start of your command along with the other parameters.

And as discussed earlier in our replies to your post, if your sample was prepared using the Nextera kit, you
need to use the adapter file for Nextera, not the TruSeq file.

[Ancestro]

This is a common error, the directory from Trimmomatic is executed is not the same as the one it was unziped. If you copied the .jar from the zip, you have to copy the illumina adapters directory too! For the error not being crucial, the trimming goes on, but you might still have the adapters in your reads (if they are not trimmed by the illumina software first).