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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.

Useful links:

First and foremost, the FastQC "Summary" should generally be ignored. Its "grading scale" (green - good, yellow - warning, red - failed) incorporates assumptions for a particular kind of experiment, and is not applicable to most real-world data. Instead, look through the individual reports and evaluate them according to your experiment type.

The FastQC reports I find most useful are:

  1. The Per base sequence quality report, which can help you decide if sequence trimming is needed before alignment.
  2. 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.
  3. The Overrepresented Sequences report, which helps evaluate adapter contamination.
Expand
titleA couple of other things to note about FastQC
  • 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,

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. Here's how to run FastQC using the version we installed:

Code Block
titleRunning FastQC example
module load fastqc

fastqc data/SRR030257_1.fastq

Exercise: FastQC results

What did FastQC create?

Expand
titleAnswer
No Format
titlels -l shows something like this
directory structure FILL IN!

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.

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:

Code Block
titleFastQC results URL
http://web.corral.tacc.utexas.edu/BioITeam/

Exercise: Should we trim this data?

Based on this FastQC output, should we trim this data?

Expand
titleAnswer

The Per base sequence quality report does not look good. The data should probably be trimmed (to 40 or 50 bp) before alignment.

Let's look at tools to do such manipulations to fastqc files, if we have to.