FASTQC and MultiQC
Contents
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.
