FASTQC and MultiQC
Assessment of short read quality. FastQC carries out analysis on a single readset, usually represented by a single fastq or fastq.gz file. MultiQC uses the output of FASTQC to aggregate the FastQC quality indicators of the different reads together, to allo inter-readset comparisons.
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/
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.
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
the dot stands for the local directory, and is obligatory.
Under its general statistics we get the following headings:
- Sample Name
- % Dups
- % GC
- M Seqs, millions of sequences
multiqc's help file