FastQC¶
Get Illumina data¶
We have prepared some Illumina data for comparison with Nanopore data for you. Get the data with:
cd ~/workdir
wget https://openstack.cebitec.uni-bielefeld.de:8080/swift/v1/coursedata2020/Illumina.tar.gz
Then unpack and remove the tar file:
cd ~/workdir/
tar -xzvf Illumina.tar.gz
rm Illumina.tar.gz
Check, what’s in the Illumina directory:
ls -l ~/workdir/Illumina/
total 109108
-rw-r--r-- 1 ubuntu ubuntu 53544266 Nov 4 14:04 D0003_S3_L001_R1_001.fastq.gz
-rw-r--r-- 1 ubuntu ubuntu 58178627 Nov 4 14:04 D0003_S3_L001_R2_001.fastq.gz
FastQC¶
FastQC aims to provide a simple way to do some quality control checks on raw sequence data coming from high throughput sequencing pipelines. It provides a modular set of analyses which you can use to give a quick impression of whether your data has any problems of which you should be aware before doing any further analysis.
The main functions of FastQC are
- Import of data from BAM, SAM or FastQ files (any variant)
- Providing a quick overview to tell you in which areas there may be problems
- Summary graphs and tables to quickly assess your data
- Export of results to an HTML based permanent report
- Offline operation to allow automated generation of reports without running the interactive application
You can run FastQC interactively or using ht CLI, which offers the following options:
fastqc --help
SYNOPSIS
fastqc seqfile1 seqfile2 .. seqfileN
fastqc [-o output dir] [--(no)extract] [-f fastq|bam|sam] [-c contaminant file] seqfile1 .. seqfileN
OPTIONS:
-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.
--nano Files come from naopore sequences and are in fast5 format. In
this mode you can pass in directories to process and the program
will take in all fast5 files within those directories and produce
a single output file from the sequences found in all files.
--nofilter If running with --casava then don't remove read flagged by
casava as poor quality when performing the QC analysis.
--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.
-k --kmers Specifies the length of Kmer to look for in the Kmer content
module. Specified Kmer length must be between 2 and 10. Default
length is 7 if not specified.
-q --quiet Supress all progress messages on stdout and only report errors.
-d --dir Selects a directory to be used for temporary files written when
generating report images. Defaults to system temp directory if
not specified.
See the FastQC home page for more info.
QA with FastQC¶
Your task is to run FastQC on the Illumina data and on your basecalled samples. Create a new directory for the results:
mkdir ~/workdir/fastqc
The results should be stored in:
~/workdir/fastqc/illumina/
and:
~/workdir/fastqc/basecall_tiny
You need to create the result directories before runnnig FastQC, because FastQC will note create them.
If you are stuck, go to the next page to get further help.
References¶
FastQC https://www.bioinformatics.babraham.ac.uk/projects/fastqc/