[simonandrews]

I've just put up v0.4.3 on our website which fixes the sequence count problem.

[flower6991]

find contaminant sequence

hello Simon,

I use FastQC to evauate my sequence data.
The last part is contaminant(overrepresented sequences)

Total Sequences 9265299
Sequence length 42

It like this:
{
>>Overrepresented sequences fail
#SequenceCountPercentagePossible Source
GATCGGAAGAGCTCGTATGCCGTCTTCTGCTTAGATCGGAAG 119288 1.2874705932317998 Illumina Single End Apapter 2 (96% over 32bp)
GATCGGAAGAGCTCGTATGCCGTCTTCTGCTTGAAAAAAAAA 112538 1.2146181143209733 Illumina Single End Apapter 2 (100% over 33bp)
AATTCGAATATCTGCCGAATGCCGTGTGGACGTAAGCGTGAA 29127 0.3143665412200945 No Hit
GATCGGAAGAGCTGTATGCCGTCTTCTGCTTAGATCGGAAGA 24460 0.2639957976531572 No Hit
AATTCACAGGTGTTCTCCCGTATTGTTGACATGCCAGCGGGT 20305 0.21915104952360417 No Hit
AATTCCCCTTGATTGCAAGGGGAACGAAATAGACAGATCGCT 17190 0.18553097962623763 No Hit
}

How can I find these contaminant sequences from all data?
use fastQC or bioperl module? or other algorithms?

Is this data's quality too poor that we can not use it to analysis ?


Thank you very much

[simonandrews]

hello Simon,

I use FastQC to evauate my sequence data.
The last part is contaminant(overrepresented sequences)

Total Sequences 9265299
Sequence length 42

It like this:
{
>>Overrepresented sequences fail
#SequenceCountPercentagePossible Source
GATCGGAAGAGCTCGTATGCCGTCTTCTGCTTAGATCGGAAG 119288 1.2874705932317998 Illumina Single End Apapter 2 (96% over 32bp)
GATCGGAAGAGCTCGTATGCCGTCTTCTGCTTGAAAAAAAAA 112538 1.2146181143209733 Illumina Single End Apapter 2 (100% over 33bp)
AATTCGAATATCTGCCGAATGCCGTGTGGACGTAAGCGTGAA 29127 0.3143665412200945 No Hit
GATCGGAAGAGCTGTATGCCGTCTTCTGCTTAGATCGGAAGA 24460 0.2639957976531572 No Hit
AATTCACAGGTGTTCTCCCGTATTGTTGACATGCCAGCGGGT 20305 0.21915104952360417 No Hit
AATTCCCCTTGATTGCAAGGGGAACGAAATAGACAGATCGCT 17190 0.18553097962623763 No Hit
}

So this is saying that you have some adapter contamination in your sample. You've probably lost 5-10% of your sequences to this contamination, but there's no reason to think that the rest of it won't be usable.

How can I find these contaminant sequences from all data?
use fastQC or bioperl module? or other algorithms?

FastQC is not intended to be a filter - merely just to report on the state of your data. There are plenty of other tools out there which you can use to remove these contaminants if you need to do that before running the rest of your analyses.

Is this data's quality too poor that we can not use it to analysis ?

There's nothing in this result to suggest that - it simply shows that the data is contaminated. You need to look at the rest of the results as well to assess the overall quality of your data.

FastQC output shouldn't be taken too literally. Just because you get a red cross against one or more tests doesn't necessarily mean that you should throw your data away. I can think of legitimate reasons why some data sets would fail every single one of the tests - and that's OK. What the program aims to do is to point things out to you ("Did you know that 3 sequences make up 50% of your data?" etc). Beyond that it's really up to you to decide if this means that the data is too poor to use, if you go ahead - but bear the FastQC results in mind in your interpretation, or if you decide the warning is spurious for the type of data you're analysing.

For example - every one of our PhiX control lanes now fails QC as assessed by FastQC because the degree of sequence duplication is ridiculously high. This is both a correct and irrelevant result. In a supposedly diverse library this would indicate a real problem, but in a PhiX lane we expect that. You have to judge the results based on your knowledge of the experiment.

[fabrice]

Fastqc: Version 0.5.0

When I run fastqc in the home directory ~/bin/FastQC, I got this error.

java -Xmx250m -classpath . uk.ac.bbsrc.babraham.FastQC.FastQCApplication


Exception in thread "main" java.lang.NoClassDefFoundError: uk/ac/bbsrc/babraham/FastQC/FastQCApplication

java version "1.5.0_17"
Java(TM) 2 Runtime Environment, Standard Edition (build 1.5.0_17-b04)
Java HotSpot(TM) 64-Bit Server VM (build 1.5.0_17-b04, mixed mode)

[simonandrews]

Fastqc: Version 0.5.0

When I run fastqc in the home directory ~/bin/FastQC, I got this error.

java -Xmx250m -classpath . uk.ac.bbsrc.babraham.FastQC.FastQCApplication


Exception in thread "main" java.lang.NoClassDefFoundError: uk/ac/bbsrc/babraham/FastQC/FastQCApplication

This will be because you have an existing classpath defined and you need to add the new directory to it, rather than replacing it.

If you're running fastqc on a unix system from the command line it's much easier to use the wrapper script which is included in the distribution.

In your case you'd initially need to do:

chmod 755 ~/bin/FastQC/fastqc

..then in future you can do:

~/bin/FastQC/fastqc [your list of files]

[fabrice]

The script fastqc does not work for command line.
On mac:
java -version
java version "1.6.0_20"
Java(TM) SE Runtime Environment (build 1.6.0_20-b02-279-10M3065)
Java HotSpot(TM) 64-Bit Server VM (build 16.3-b01-279, mixed mode)

./fastqc aa.txt
Exception in thread "main" java.lang.NoClassDefFoundError: uk/ac/bbsrc/babraham/FastQC/FastQCApplication
Caused by: java.lang.ClassNotFoundException: uk.ac.bbsrc.babraham.FastQC.FastQCApplication
at java.net.URLClassLoader$1.run(URLClassLoader.java:202)
at java.security.AccessController.doPrivileged(Native Method)
at java.net.URLClassLoader.findClass(URLClassLoader.java:190)
at java.lang.ClassLoader.loadClass(ClassLoader.java:307)
at sun.misc.Launcher$AppClassLoader.loadClass(Launcher.java:301)
at java.lang.ClassLoader.loadClass(ClassLoader.java:248)

On debian:

java -version
java version "1.5.0"
gij (GNU libgcj) version 4.3.2

Copyright (C) 2007 Free Software Foundation, Inc.
This is free software; see the source for copying conditions. There is NO
warranty; not even for MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE.

./fastqc a.txt
Exception in thread "main" java.lang.NoClassDefFoundError: uk.ac.bbsrc.babraham.FastQC.FastQCApplication
at gnu.java.lang.MainThread.run(libgcj.so.90)
Caused by: java.lang.ClassNotFoundException: uk.ac.bbsrc.babraham.FastQC.FastQCApplication not found in gnu.gcj.runtime.SystemClassLoader{urls=[file:./,file:~/bin/FastQC/,file:~/bin/FastQC/], parent=gnu.gcj.runtime.ExtensionClassLoader{urls=[], parent=null}}
at java.net.URLClassLoader.findClass(libgcj.so.90)
at java.lang.ClassLoader.loadClass(libgcj.so.90)
at java.lang.ClassLoader.loadClass(libgcj.so.90)
at gnu.java.lang.MainThread.run(libgcj.so.90)

This will be because you have an existing classpath defined and you need to add the new directory to it, rather than replacing it.

If you're running fastqc on a unix system from the command line it's much easier to use the wrapper script which is included in the distribution.

In your case you'd initially need to do:

chmod 755 ~/bin/FastQC/fastqc

..then in future you can do:

~/bin/FastQC/fastqc [your list of files]

[fabrice]

On unbantu:

java -version
java version "1.6.0_18"
Java(TM) SE Runtime Environment (build 1.6.0_18-b07)
Java HotSpot(TM) 64-Bit Server VM (build 16.0-b13, mixed mode)

Exception in thread "main" java.lang.NoClassDefFoundError: uk/ac/bbsrc/babraham/FastQC/FastQCApplication
Caused by: java.lang.ClassNotFoundException: uk.ac.bbsrc.babraham.FastQC.FastQCApplication
at java.net.URLClassLoader$1.run(URLClassLoader.java:202)
at java.security.AccessController.doPrivileged(Native Method)
at java.net.URLClassLoader.findClass(URLClassLoader.java:190)
at java.lang.ClassLoader.loadClass(ClassLoader.java:307)
at sun.misc.Launcher$AppClassLoader.loadClass(Launcher.java:301)
at java.lang.ClassLoader.loadClass(ClassLoader.java:248)
Could not find the main class: uk.ac.bbsrc.babraham.FastQC.FastQCApplication. Program will exit.

[simonandrews]

Exception in thread "main" java.lang.NoClassDefFoundError: uk/ac/bbsrc/babraham/FastQC/FastQCApplication

Could you by any chance have downloaded the source distribution instead of the compiled version? The errors are all saying that java can't find the initial class file, which it should be able to if the classpath is set correctly.

Can you look in uk/ac/bbsrc/babraham/FastQC/ and see if you see a file called FastQCApplication.class. If you see a file called FastQCApplication.java then you've got the source files rather than the binaries.

[fabrice]

The files are:

Analysis FastQCApplication.java Graphs Modules Resources Sequence
Dialogs FastQCMenuBar.java Help Report Results Statistics

Could you by any chance have downloaded the source distribution instead of the compiled version? The errors are all saying that java can't find the initial class file, which it should be able to if the classpath is set correctly.

Can you look in uk/ac/bbsrc/babraham/FastQC/ and see if you see a file called FastQCApplication.class. If you see a file called FastQCApplication.java then you've got the source files rather than the binaries.

[simonandrews]

The files are:
FastQCApplication.java

Those are the source code files (which is why it won't run). You need to download the compiled version, either the generic zip file or the Mac application bundle.

[simonandrews]

FastQC v0.5.1 has been released. This fixes a formatting bug in the text output and a bug in the %GC profile for runs containing reads >100bp.

We've also improved the fitting of the modelled curve to the %GC profile and have added a load more oligos to the contaminants file (thanks to Aaron Statham for providing these).

You can get the new version from:

http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/

[If you don't see the new version of any page hit shift+refresh to force our cache to update]

[simonandrews]

I should add that for announcements of new releases to all of the software from Babraham (FastQC, SeqMonk, Bismark etc) you can now follow us on twitter at @babraham_bioinf. I'll still post more complete announcements here though.

[seq_GA]

Hi Simon,
May I know how to use the tool in linux environment? Thanks.

[simonandrews]

Hi Simon,
May I know how to use the tool in linux environment? Thanks.

Instructions for installing and running the program on a variety of platforms are in the INSTALL.txt document which comes in the distribution. On linux there is a wrapper script which you can use to run the program which is probably the easiest way to launch it.

[seq_GA]

Hi Simon,
Thanks for your response. I am trying to use this as part of the pipeline and hence didn't try it through win32 to access the linux server.

I tried as below and please let me know the details.

FastQC]$ chmod 755 fastqc
FastQC]$ ./fastqc 
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:173)
        at java.awt.Window.<init>(Window.java:437)
        at java.awt.Frame.<init>(Frame.java:419)
        at java.awt.Frame.<init>(Frame.java:384)
        at javax.swing.JFrame.<init>(JFrame.java:180)
        at uk.ac.bbsrc.babraham.FastQC.FastQCApplication.<init>(FastQCApplication.java:256)
        at uk.ac.bbsrc.babraham.FastQC.FastQCApplication.main(FastQCApplication.java:91)

Thanks.