Contents
How do I read FastQC output?
The output from FastQC, after analyzing a FASTQ file of sequence reads, is an html file that may be viewed in your browser. The report contains one result section for each FastQC module. In addition to the graphical or list data provided by each module, a flag of “Passed”, “Warn” or “Fail” is assigned.
Why is FastQC important?
FastQC provides 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.
What is per base GC content?
Summary. This module measures the GC content across the whole length of each sequence in a file and compares it to a modelled normal distribution of GC content. A normal distribution which is shifted indicates some systematic bias which is independent of base position.
How do I run FastQC?
Actually installing FastQC is as simple as unzipping the zip file it comes in into a suitable location. That’s it. Once unzipped it’s ready to go. You can run FastQC in one of two modes, either as an interactive graphical application in which you can dynamically load FastQ files and view their results.
How can I check the quality of Fastq files?
Run FASTQ Quality Check This functionality can be found under Tools → FASTQ Tools → FASTQ Quality Check.
What is considered high GC content?
Above 60% is considered high GC and therefore Actinobacteria, and below 60% is considered low, and therefore Firmicute. The GC content can help in the demarcation of bacterial species.
What is considered GC rich?
Thank you for your clear answer! It is my understanding that when using the GC content as a way to classify a bacteria into either the Firmicute or Actinobacteria phylum 60% is the cutoff. Above 60% is considered high GC and therefore Actinobacteria, and below 60% is considered low, and therefore Firmicute.
Why does FastQC show unexpectedly high sequence levels?
As a consequence FASTQC tends to generate unnerving warnings for multiple Illumina sequencing data types; This often includes unnecessary warnings about sequence duplication levels and overrepresented sequences. More modern tools like HTStream and FASTP use more sophisticated algorithms for these purposes and also work on paired-end read data.
How does the FastQC table help identify contamination?
This table aids in identifying contamination, such as vector or adapter sequences. If the %GC content was off in the above module, this table can help identify the source. If not listed as a known adapter or vector, it can help to BLAST the sequence to determine the identity.
Which is the most important module in FastQC?
One of the most important analysis modules is the “Per base sequence quality” plot. This plot provides the distribution of quality scores at each position in the read across all reads. This plot can alert us to whether there were any problems occuring during sequencing and whether we might need to contact the sequencing facility.
How are base positions binned together in FastQC?
The number of base positions binned together depends on the length of the read; for example, with 150bp reads the latter part of the plot will report aggregate statistics for 5bp windows. Shorter reads will have smaller windows and longer reads larger windows.