[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]

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

[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]

[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

[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 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 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]

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,

[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]

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!

[GenoMax]

repair.sh from BBTools will do that easily: http://seqanswers.com/forums/showpos...8&postcount=61

[drdna]

Thanks for the insights. BaseSpace does a good job of adaptor trimming, demultiplexing. It's probably more efficient to just run the reads through a script to pair them up properly before downstream processing.

[drdna]

That's probably it - we use BaseSpace. My guess is that BaseSpace is filtering out pairs with poor quality. I'll have to look into that.

[GenoMax]

Only way I would see that happen is you get consistently bad quality reads on one end that are removed by MiSeq reporter/BaseSpace. Perhaps you should ask the tech to turn off on-instrument analysis (adapter trimming etc) and you can do that offline.

[westerman]

Settings would be in the sample sheet.

Are you getting the reads directly from the sequencer or via BaseSpace? The latter may be doing some sort of trimming for you. Because we have a hiSeq and pre-existing pipelines we do not use BaseSpace but rather just grab the raw reads. Thus I am not familiar with BaseSpace but I do know that it can do a lot of useful processing.