FASTQC and MultiQC

Introduction

Assessment of short read quality. FastQC carries out analysis on a single readsets, usually represented by a single fastq or fastq.gz file. It is of no concern whether the reads come from single or paired libraries. FastQC calls these sequences, rather than reads.

MultiQC uses the output of FASTQC to aggregate the FastQC quality indicators of the different reads together, to allo inter-readset comparisons.

FastQC

Available on cluster via the module load FASTQC command.

This is a very widely used program which however, is not based on a publication. It is a software release.

It gives quite detailed graphs on the nature of the readset, and details to the different graphics can be found at the following link: http://www.bioinformatics.babraham.ac.uk/projects/fastqc/Help/3%20Analysis%20Modules/

FastQC's modules

Each graph and accompanying analysis is called a module. The principal graph is that of per base quality. The documentation says:

• The central red line is the median value
• The yellow box represents the inter-quartile range (25-75%)
• The upper and lower whiskers represent the 10% and 90% points
• The blue line represents the mean quality

FastQC's help file

SYNOPSIS

   fastqc seqfile1 seqfile2 .. seqfileN

   fastqc [-o output dir] [--(no)extract] [-f fastq|bam|sam] [-c contaminant file] seqfile1 .. seqfileN

DESCRIPTION

   FastQC reads a set of sequence files and produces from each one a quality
   control report consisting of a number of different modules, each one of 
   which will help to identify a different potential type of problem in your
   data.
   
   If no files to process are specified on the command line then the program
   will start as an interactive graphical application.  If files are provided
   on the command line then the program will run with no user interaction
   required.  In this mode it is suitable for inclusion into a standardised
   analysis pipeline.
   
   The options for the program as as follows:
   
   -h --help       Print this help file and exit
   
   -v --version    Print the version of the program and exit
   
   -o --outdir     Create all output files in the specified output directory.
                   Please note that this directory must exist as the program
                   will not create it.  If this option is not set then the 
                   output file for each sequence file is created in the same
                   directory as the sequence file which was processed.
                   
   --casava        Files come from raw casava output. Files in the same sample
                   group (differing only by the group number) will be analysed
                   as a set rather than individually. Sequences with the filter
                   flag set in the header will be excluded from the analysis.
                   Files must have the same names given to them by casava
                   (including being gzipped and ending with .gz) otherwise they
                   won't be grouped together correctly.
                   
   --nofilter      If running with --casava then don't remove read flagged by
                   casava as poor quality when performing the QC analysis.
                  
   --extract       If set then the zipped output file will be uncompressed in
                   the same directory after it has been created.  By default
                   this option will be set if fastqc is run in non-interactive
                   mode.
                   
   -j --java       Provides the full path to the java binary you want to use to
                   launch fastqc. If not supplied then java is assumed to be in
                   your path.
                  
   --noextract     Do not uncompress the output file after creating it.  You
                   should set this option if you do not wish to uncompress
                   the output when running in non-interactive mode.
                   
   --nogroup       Disable grouping of bases for reads >50bp. All reports will
                   show data for every base in the read.  WARNING: Using this
                   option will cause fastqc to crash and burn if you use it on
                   really long reads, and your plots may end up a ridiculous size.
                   You have been warned!
                   
   -f --format     Bypasses the normal sequence file format detection and
                   forces the program to use the specified format.  Valid
                   formats are bam,sam,bam_mapped,sam_mapped and fastq
                   
   -t --threads    Specifies the number of files which can be processed
                   simultaneously.  Each thread will be allocated 250MB of
                   memory so you shouldn't run more threads than your
                   available memory will cope with, and not more than
                   6 threads on a 32 bit machine
                 
   -c              Specifies a non-default file which contains the list of
   --contaminants  contaminants to screen overrepresented sequences against.
                   The file must contain sets of named contaminants in the
                   form name[tab]sequence.  Lines prefixed with a hash will
                   be ignored.

   -a              Specifies a non-default file which contains the list of
   --adapters      adapter sequences which will be explicity searched against
                   the library. The file must contain sets of named adapters
                   in the form name[tab]sequence.  Lines prefixed with a hash
                   will be ignored.
                   
   -l              Specifies a non-default file which contains a set of criteria
   --limits        which will be used to determine the warn/error limits for the
                   various modules.  This file can also be used to selectively 
                   remove some modules from the output all together.  The format
                   needs to mirror the default limits.txt file found in the
                   Configuration folder.

MultiQC

A relatively new tool that aggregates the output of FASTQC into one report.

• available on the command line with any module loading as it is a python module (already installed easily via pip)

Go into the directory where the FASTQC output is and run

multiqc .

the dot stands for the local directory, and is obligatory.

Under its general statistics we get the following headings:

• Sample Name
•  % Dups
•  % GC
• Length
• M Seqs, millions of sequences

multiqc's help file

Usage: multiqc [OPTIONS] <analysis directory>

  MultiQC aggregates results from bioinformatics analyses across many
  samples into a single report.

  It searches a given directory for analysis logs and compiles a HTML
  report. It's a general use tool, perfect for summarising the output from
  numerous bioinformatics tools.

  To run, supply with one or more directory to scan for analysis results.
  To run here, use 'multiqc .'

  See http://multiqc.info for more details.

  Author: Phil Ewels (http://phil.ewels.co.uk)

Options:
  -f, --force                     Overwrite any existing reports
  -d, --dirs                      Prepend directory to sample names
  -s, --fullnames                 Do not clean the sample names (leave as full
                                  file name)
  -i, --title TEXT                Report title. Printed as page header, used
                                  for filename if not otherwise specified.
  -n, --filename TEXT             Report filename. Use 'stdout' to print to
                                  standard out.
  -o, --outdir TEXT               Create report in the specified output
                                  directory.
  -t, --template [default|default_dev|geo|simple]
                                  Report template to use.
  -x, --ignore TEXT               Ignore analysis files (glob expression)
  -e, --exclude [module name]     Do not use this module. Can specify multiple
                                  times.
  -m, --module [module name]      Use only this module. Can specify multiple
                                  times.
  --data-dir / --no-data-dir      Specify whether the parsed data directory
                                  should be created.
  -k, --data-format [tsv|yaml|json]
                                  Output parsed data in a different format
  -z, --zip-data-dir              Compress the data directory.
  --flat                          Use only flat plots (static images)
  --interactive                   Use only interactive plots (HighCharts
                                  Javascript)
  -c, --config PATH               Specific config file to load, after those in
                                  MultiQC dir / home dir / working dir.
  -v, --verbose                   Increase output verbosity.
  -q, --quiet                     Only show log warnings
  --version                       Show the version and exit.
  -h, --help                      Show this message and exit.