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| title | A couple of other things to note about FastQC |
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- For many of its reports, FastQC analyzes only the first 200,000 sequences in order to keep processing and memory requirements down.
- Some of FastQC's graphs have a 1-100 vertical scale that is tricky to interpret. The 100 is a relative marker for the rest of the graph. For example, sequence duplication levels are relative to the number of unique sequences,
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Running FastQC
FastQC is not currently available from the TACC module system, but the command-line version has been installed in the $BI/bin/FastQC directory (downloaded from the Babraham Bioinformatics web site; interactive GUI versions are also available for Windows and Macintosh).FastQC creates a sub-directory for each analyzed FASTQ file, so we should copy the file we want to look at locally first. iis available on lonestar as a module.
Here's how to run FastQC using the version we installedon our sample data:
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| title | Running FastQC example |
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module load fastqc
fastqc data/SRR030257Sample1_1R1.fastq |
Exercise: FastQC results
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| title | ls -l shows something like this |
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| directory structure FILL IN!
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The Sample_Yeast_L005_R1.cat.fastq.gz file is what we analyzed, so FastQC created the other two items. Sample_Yeast_L005_R1.cat_fastqc is a directory (the "d" in "drwxrwxr-x"), so use ls Sample_Yeast_L005_R1.cat_fastqc to see what's in it. Sample_Yeast_L005_R1.cat_fastqc.zip is just a Zipped (compressed) version of the whole directory.drwxrwx--- 4 daras G-801020 32768 May 16 14:03 Sample1_R1_fastqc
-rw-rw---- 1 daras G-801020 186116 May 16 13:58 Sample1_R1_fastqc.zip |
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Looking at FastQC output
You can't run a web browser directly from your "dumb terminal" command line environment. The FastQC results have to be placed where a web browser can access them. We put a copy at this URL:
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http://web.corral.tacc.utexas.edu/BioITeam/
/rnaseq_course/fastqc_exercise/Sample1_R1_fastqc/fastqc_report.html |
Exercise: Should we trim this data?
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The Per base sequence quality report does not look good. The data should probably be trimmed (to 40 or 50 bp) before alignmentshows that trimming the las 20 bp or so would not be a bad idea. |
Let's look at tools to do such manipulations to fastqc files, if we have to.