The first order of business after receiving sequencing data should be to check your data quality. This often-overlooked step helps guide the manner in which you process the data, and can prevent many headaches.
FastQC
FastQC is a tool that produces a quality analysis report on FASTQ files.
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- The Per base sequence quality report, which can help you decide if sequence trimming is needed before alignment.
2. The Per Sequence Quality Score report, which can tell you if a subset of your reads just have poor quality scores. These reads can be completely filtered from analysis.
3. The Sequence Duplication Levels report, which helps you evaluate library enrichment / complexity. But note that different experiment types are expected to have vastly different duplication profiles.
- The Overrepresented Sequences report, which helps evaluate adapter contamination.
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| title | A couple of other things to note about FastQC |
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Note: For many of its reports, FastQC analyzes only the first 200,000 sequences in order to keep processing and memory requirements down.
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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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module load fastqc fastqc data/SRR030257Sample1_1R1.fastq |
Exercise: FastQC results
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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 last 10 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.