[GenoMax]

At a minimum did you make sure that full file paths are included to wherever these trimmed files are when you ran SoapdeNovo2? The error message seems to indicate that these files can't be opened/read. Also did you make sure that these files actually have something in them i.e. they are not empty (I know it sounds stupid but this is a problem new people sometimes have with unix files).

[mgbfx9]

Hi,

I got another problem, I need your help.

I used Trimmomatic-0.33 to preprocess my MiSeq data which removed my adapter sequences and the low-quality reads and generated fastq files.
Now I took this processed fastq files to do de novo assembly with Soapdenovo2, the assembler could not open my file. It has the same header as the original. Output says that "Import reads from file...... Cannot open. Now exiting the system".

I used Soapdenovo2 for de novo assembly of these MiSeq data before preprocessing, they could read my file, but as the contig values were pretty low, I preprocessed my data, but now the assembler could not open the preprocessed files.

What could be wrong?
Any help would be appreciated, thanks!

[mgbfx9]

Finally, I got the problem solved. That is not due to the space in the code, but a small thing, -trimlog was –trimlog! I can not believe that minor thing can make so much problem.

Thank you everyone for all the help.

[mgbfx9]

Hi kmcarr,
Thank you for your response. Let me check again my codes, and will let you know the update.

[kmcarr]

Hi kmcarr and mastal,

Thank you for your suggestion.

No, I didn't have any spaces in between the original code. Here when I posted my code, I took out the name of my directory for privacy, so here I missed the spaces when I was typing. Although I checked again my code and ran, but got the same message. However, I am not sure whether I have missing spaces in places where they should not be. I can give you the code again.

Thanks in advance for your suggestion,

Exception in thread "main" java.lang.RuntimeException: [COLOR="Red"]Unknown trimmer: /home/mydir/ngs_trimm/output_reverseP.fastq[/COLOR]

Trimmomatic clearly states what error it encountered. The only reasonable explantation for this error is that you have inadvertently passed extra arguments to Trimmomatic on your command line, and the presence of spaces in you pathnames as I outlined above would lead to the creation of these extra arguments and Trimmomatic throwing exactly the error it has.

[mgbfx9]

Hi kmcarr and mastal,

Thank you for your suggestion.

No, I didn't have any spaces in between the original code. Here when I posted my code, I took out the name of my directory for privacy, so here I missed the spaces when I was typing. Although I checked again my code and ran, but got the same message. However, I am not sure whether I have missing spaces in places where they should not be. I can give you the code again.

Thanks in advance for your suggestion,

[mastal]

Have you checked to make sure the error isn't caused by having spaces in the wrong places (in the middle of the path to a file), or missing spaces in places where there should be spaces?

In the code that you have posted above, for example, there are spaces between 'home' and the rest of your path in at least a couple of places.

[kmcarr]

Thank you GenoMax, mastal, Ancestro for your suggestions. I was not well, so couldn’t do further and I'm sorry for my delayed response of your help.

I took all of your suggestions, and if I understood correctly, I modified the code the following way, and ran from the directory where the Trimmomatic is located, but I think the program couldn’t find the trimmer.

[mydir Trimmomatic-0.33]$ java -jar trimmomatic-0.33.jar PE -threads 8 -phred33 –trimlog logfile_sample1 /home/mydir/ngs_trimm/sample1_L001_R1_001.fastq /home/[COLOR="Red"]mydir /ngs_trimm[/COLOR]/sample1_L001_R2_001.fastq /home/mydir/ngs_trimm/output_forwardP.fastq /home[COLOR="Red"]/ mydir/[/COLOR]ngs_trimm/output_forwardU.fastq /home/mydir/ngs_trimm/output_reverseP.fastq /home[COLOR="red"]/ mydir/[/COLOR]ngs_trimm/output_reverseU.fastq ILLUMINACLIP:/home/mydir/Trimmomatic-0.33/adapters/NexteraPE-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36     

Output:
TrimmomaticPE: Started with arguments: -threads 8 -phred33 –trimlog /home/mydir/ngs_trimm/logfile_sample1 ……. Started with my full code and
Exception in thread "main" java.lang.RuntimeException: Unknown trimmer: /home/mydir/ngs_trimm/output_reverseP.fastq
        at org.usadellab.trimmomatic.trim.TrimmerFactory.makeTrimmer(TrimmerFactory.java:60)
        at org.usadellab.trimmomatic.TrimmomaticPE.run(TrimmomaticPE.java:496)
        at org.usadellab.trimmomatic.Trimmomatic.main(Trimmomatic.java:35)

What did I do wrong? Any help is appreciated!
Thank you in advance,

What went wrong is you appear to have a number of spaces (three in total) in your command line that shouldn't be there. See the portions of your command line above which I have highlighted in red (BTW it makes command line and code segments much easier to read on this forum if you use the 'CODE' tags to surround them when creating post.)

Trimmomatic, like many *NIX commands interprets groups separated by spaces on a command line to be separate arguments. With the improper spaces in your command here is how Trimmomatic is interpreting what you typed:

Input file #1:           /home/mydir/ngs_trimm/sample1_L001_R1_001.fastq
Input file #2:           /home/mydir
Output file #1 paired:   /ngs_trimm/sample1_L001_R2_001.fastq
Output file #1 unpaired: /home/mydir/ngs_trimm/output_forwardP.fastq
Output file #2 paired:   /home/
Output file #2 unpaired: mydir/ngs_trimm/output_forwardU.fastq
First trimmer to apply:  /home/mydir/ngs_trimm/output_reverseP.fastq
etc., etc.

Trimmomatic is telling you in the error message that it doesn't recognize the trimmer "/home/mydir/ngs_trimm/output_reverseP.fastq" (obviously). Remove the improper spaces from your command line and try again.

[mgbfx9]

Thank you GenoMax, mastal, Ancestro for your suggestions. I was not well, so couldn’t do further and I'm sorry for my delayed response of your help.

I took all of your suggestions, and if I understood correctly, I modified the code the following way, and ran from the directory where the Trimmomatic is located, but I think the program couldn’t find the trimmer.

[mydir Trimmomatic-0.33]$ java -jar trimmomatic-0.33.jar PE -threads 8 -phred33 –trimlog logfile_sample1 /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:/home/mydir/Trimmomatic-0.33/adapters/NexteraPE-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36

Output:
TrimmomaticPE: Started with arguments: -threads 8 -phred33 –trimlog /home/mydir/ngs_trimm/logfile_sample1 ……. Started with my full code and
Exception in thread "main" java.lang.RuntimeException: Unknown trimmer: /home/mydir/ngs_trimm/output_reverseP.fastq
at org.usadellab.trimmomatic.trim.TrimmerFactory.makeTrimmer(TrimmerFactory.java:60)
at org.usadellab.trimmomatic.TrimmomaticPE.run(TrimmomaticPE.java:496)
at org.usadellab.trimmomatic.Trimmomatic.main(Trimmomatic.java:35)

What did I do wrong? Any help is appreciated!
Thank you in advance,

[Ancestro]

Btw, I had a weird issue in trimmomatic last day, it confuses files from the options or the trimming commands, i checked for additional spaces or strange characters, but nothing. Later I retyped the command as the first time and worked again. Do someone had a same problem?

[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).

[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.

[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

[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]

Perfect. Things should go much better now.