FastQC: A quality control application for FastQ data

[ksw9]

Hi,
I am new to NGS bioinformatics and am trying to use Fastqc on Illumina paired end reads. I keep getting the error:

Exception in thread "main" java.lang.NoClassDefFoundError: uk.ac.babraham.FastQC.FastQCApplication
at java.lang.Class.initializeClass(libgcj.so.10)
Caused by: java.lang.ClassFormatError: uk.ac.babraham.FastQC.Modules.QCModule (erroneous constant pool tag)


Any insight would be very helpful. Thank you in advance for your help!

[mastal]

What version of FastQC are you using,

and what is the command you used to run FastQC?

[simonandrews]

This looks like the download you have is corrupted. That error is an internal error in the validation of the class files.

I suspect if you go back and re-download and extract the fastqc install again it will fix itself.

[ksw9]

Thank you for all the help! You're right the download was corrupted.

[le.nono]

Hello there

I can't solve that problem by myself I have this error , I have no idea to solve it. I would need some good advice. Here it is below
Thx.

nono@nono-VGN-NS31M-W:~/FastQC$ fastqc ~/bioinfo/ngs/exercice_edgeR/reads/Galaxy5-\[brain_2.fastq\].fa
Started analysis of Galaxy5-[brain_2.fastq].fa
Approx 5% complete for Galaxy5-[brain_2.fastq].fa
Approx 10% complete for Galaxy5-[brain_2.fastq].fa
Approx 15% complete for Galaxy5-[brain_2.fastq].fa
Approx 20% complete for Galaxy5-[brain_2.fastq].fa
Approx 25% complete for Galaxy5-[brain_2.fastq].fa
Approx 30% complete for Galaxy5-[brain_2.fastq].fa
Approx 35% complete for Galaxy5-[brain_2.fastq].fa
Approx 40% complete for Galaxy5-[brain_2.fastq].fa
Approx 45% complete for Galaxy5-[brain_2.fastq].fa
Approx 50% complete for Galaxy5-[brain_2.fastq].fa
Approx 55% complete for Galaxy5-[brain_2.fastq].fa
Approx 60% complete for Galaxy5-[brain_2.fastq].fa
Approx 65% complete for Galaxy5-[brain_2.fastq].fa
Approx 70% complete for Galaxy5-[brain_2.fastq].fa
Approx 75% complete for Galaxy5-[brain_2.fastq].fa
Approx 80% complete for Galaxy5-[brain_2.fastq].fa
Approx 85% complete for Galaxy5-[brain_2.fastq].fa
Approx 90% complete for Galaxy5-[brain_2.fastq].fa
Approx 95% complete for Galaxy5-[brain_2.fastq].fa
Approx 100% complete for Galaxy5-[brain_2.fastq].fa
Analysis complete for Galaxy5-[brain_2.fastq].fa
Failed to process file Galaxy5-[brain_2.fastq].fa
java.lang.NullPointerException
at uk.ac.babraham.FastQC.Report.HTMLReportArchive.startDocument(HTMLReportArchive.java:170)
at uk.ac.babraham.FastQC.Report.HTMLReportArchive.<init>(HTMLReportArchive.java:60)
at uk.ac.babraham.FastQC.Analysis.OfflineRunner.analysisComplete(OfflineRunner.java:157)
at uk.ac.babraham.FastQC.Analysis.AnalysisRunner.run(AnalysisRunner.java:108)
at java.lang.Thread.run(Thread.java:744)

[santhilalsubhash]

Originally Posted by shurjo

Hi Simon,

I would really like to use FastQC for my project but am getting the following error message when I try to run it non-interactively on our Linux cluster:

$ java -Xmx250m -cp ~/bin/fastqc/FastQC

uk.ac.bbsrc.babraham.FastQC.FastQCApplication testFastQC.fastq
Exception in thread "main" java.awt.HeadlessException:
No X11 DISPLAY variable was set, but this program performed an operation which requires it.
at java.awt.GraphicsEnvironment.checkHeadless(GraphicsEnvironment.java:159)
at java.awt.Window.<init>(Window.java:431)
at java.awt.Frame.<init>(Frame.java:403)
at java.awt.Frame.<init>(Frame.java:368)
at javax.swing.JFrame.<init>(JFrame.java:158)
at uk.ac.bbsrc.babraham.FastQC.FastQCApplication.<init>(FastQCApplication.java:197)
at uk.ac.bbsrc.babraham.FastQC.FastQCApplication.main(FastQCApplication.java:63)

These are the details for our java installation:

[sensh@saturn FastQC]$ java -version
java version "1.6.0_17"
Java(TM) SE Runtime Environment (build 1.6.0_17-b04)
Java HotSpot(TM) 64-Bit Server VM (build 14.3-b01, mixed mode)

Any pointers?

Thanks,

Shurjo

I know this is basic and sorry if you would have tried... But if you have SSH server, try to connect with this

ssh -XY user@yourserver

And try to open your graphical programs with terminal.

[le.nono]

Thank you but it didn't help. The error is different I don't have a graphical problem, it's more about a class that java doesn't find when it wants to create the HTML fastqc file. Anybody help ?

[simonandrews]

I had a look in the code. That error suggests that you might have deleted or moved the Templates/Icons directory in the fastqc install. You are free to add more stuff into the templates directory to customise your reports but you have to leave the icons which come with the program in place.

[simonandrews]

I've just seen that there are two conversations which seem to be getting conflated here. Just to be clear, it's le.nono who has the problem with the templates directory, and shurjo who is trying to run FastQC interactively in a unix session without an X tunnel set up.

[le.nono]

Got it thank you Simon!

[le.nono]

Well I've reinstall fastqc, still have the same error. I've tried on Galaxy, with the same file it works fine...

[le.nono]

My problem is solved thank you! Your comment helped me to investigate, and came from my user Ubuntu access rights.

[F_KVH]

Hi,
I have installed FastQC on OSX 10.9.1 and have java version java version "1.6.0_65"
Java(TM) SE Runtime Environment (build 1.6.0_65-b14-462-11M4609)
Java HotSpot(TM) 64-Bit Server VM (build 20.65-b04-462, mixed mode)

but when I run FastQC, I get error:
Exception in thread "main" java.lang.NoClassDefFoundError: uk/ac/babraham/FastQC/FastQCApplication
Caused by: java.lang.ClassNotFoundException: uk.ac.babraham.FastQC.FastQCApplication
at java.net.URLClassLoader$1.run(URLClassLoader.java:202)
at java.security.AccessController.doPrivileged(Native Method)
at java.net.URLClassLoader.findClass(URLClassLoader.java:190)
at java.lang.ClassLoader.loadClass(ClassLoader.java:306)
at sun.misc.Launcher$AppClassLoader.loadClass(Launcher.java:301)
at java.lang.ClassLoader.loadClass(ClassLoader.java:247)
Any suggession to solve it?
Thanks

[simonandrews]

Originally Posted by F_KVH

Hi,
I have installed FastQC on OSX 10.9.1 and have java version java version "1.6.0_65"
Java(TM) SE Runtime Environment (build 1.6.0_65-b14-462-11M4609)
Java HotSpot(TM) 64-Bit Server VM (build 20.65-b04-462, mixed mode)

but when I run FastQC, I get error:
Exception in thread "main" java.lang.NoClassDefFoundError: uk/ac/babraham/FastQC/FastQCApplication
Caused by: java.lang.ClassNotFoundException: uk.ac.babraham.FastQC.FastQCApplication
at java.net.URLClassLoader$1.run(URLClassLoader.java:202)
at java.security.AccessController.doPrivileged(Native Method)
at java.net.URLClassLoader.findClass(URLClassLoader.java:190)
at java.lang.ClassLoader.loadClass(ClassLoader.java:306)
at sun.misc.Launcher$AppClassLoader.loadClass(Launcher.java:301)
at java.lang.ClassLoader.loadClass(ClassLoader.java:247)
Any suggession to solve it?
Thanks

This error suggests that the program isn't able to find the installation folder correctly to find its other resources. There are a number of possible reasons for this included corrupted downloads or mis-extracted zip files, but the most common cause we've seen is that the fastqc program has been copied out of the installation folder to another location (often /usr/local/bin/ so it ends up in the path). If you want to add fastqc to your path then you need to link (using ln -s ) rather than copy the fastqc launcher program into your path.

A quick check to see if this is the case would be to run

which fastqc

The location returned should either be the fastqc program inside the full installation folder, or a link to that file. If it's anything else then that will be why the launcher is failing.

If it's not that then I'd suggest re-downloading and extracting the software as it could be that there are simply files missing from the installation.

[F_KVH]

Dear Simon,
Thanks for your help. I installed again & now it works. Do you think I need to install xcode app?
Best regards

[simonandrews]

Originally Posted by F_KVH

Dear Simon,
Thanks for your help. I installed again & now it works. Do you think I need to install xcode app?
Best regards

No, you'd only need something like xcode if you wanted to edit the source code. It wouldn't be needed just to run it.

[boilermaker]

Originally Posted by lletourn

The illumina RNA protocol uses random hexamers to amplify the RNA. The thing is they are not 100% random so the beginning looks skewed for base composition, but that's because of the amplification.

For mapping it's no problem. For assembly it might confuse some assemblers. (When assembling I would trim the 5' of RNA, not for mapping)

Our group recently used the HiSeq 2000 platorm to generate transcriptome data (single-end, 50 bp reads). I have noticed that Illumina transcriptome sequencing yields typically yield these errors ("Per base sequence content" and "Per base GC content") during FastQC analysis. You suggest that these can safely be ignored when mapping to a genome? I wasn't sure if there was a "best practices" approach to dealing with these biases. Is there a mapping algorithm that is preferred among those who are dealing with Illumina transcriptome sequencing data?

(I am sorry if this question has been answered thoroughly elsewhere in the forum... I have only just joined, and despite trying to navigate the posts with the "Search" tool, I have not yet come across an answer).

[Brian Bushnell]

Originally Posted by boilermaker

Our group recently used the HiSeq 2000 platorm to generate transcriptome data (single-end, 50 bp reads). I have noticed that Illumina transcriptome sequencing yields typically yield these errors ("Per base sequence content" and "Per base GC content") during FastQC analysis. You suggest that these can safely be ignored when mapping to a genome? I wasn't sure if there was a "best practices" approach to dealing with these biases.

It would probably be better to ignore them than try to correct them, though if you posted the fastqc graphs it would be easier to say.

Is there a mapping algorithm that is preferred among those who are dealing with Illumina transcriptome sequencing data?

I prefer BBMap when mapping Illumina RNA-seq data. It's more robust to errors than other RNA-seq aligners, and doesn't require an annotation file. Oh, and I wrote it, but that's not why.

[boilermaker]

Originally Posted by Brian Bushnell

It would probably be better to ignore them than try to correct them, though if you posted the fastqc graphs it would be easier to say.

Thank you Brian. I will certainly give BBMap a try (and thank you very much for scripting it!)

I have attached a "typical" fastqc graphs (per base gc content, per base sequence content) from one of my datasets (most have profiles like this example).

[Brian Bushnell]

Doesn't look ideal, but I can't think of a good way to improve it, assuming you've already trimmed adapters (which can alter the base composition).