Contents
What is KMER content in FastQC?
Kmer Content Measures the count of each short nucleotide of length k (default = 7) starting at each positon along the read. Any given Kmer should be evenly represented across the length of the read. A list of kmers which appear at specific positions with greater than expected frequency are reported.
What is per base sequence quality?
Per base sequence quality. It shows an overview of the range of quality, measured as phred scores across all bases at each position in the FastQ file (raw reads). It measures the GC content across the whole length of each sequence in a raw read file and compares it to a modelled normal distribution of GC content.
What is sequence quality?
What is a Quality Score in Sequencing? Sequencing quality scores measure the probability that a base is called incorrectly. With sequencing by synthesis (SBS) technology, each base in a read is assigned a quality score by a phred-like algorithm1,2, similar to that originally developed for Sanger sequencing experiments.
How much DNA is needed for Illumina sequencing?
If you have prepared your own Illumina compatible sequencing libraries, then we require at least 10 nM sample DNA in 10 μl. If you require sample de-multiplexing please provide us with the index sequences used.
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 overrepresented sequences used in FastQC?
The “Overrepresented sequences” table is another important module as it displays the sequences (at least 20 bp) that occur in more than 0.1% of the total number of sequences. 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.
How is the per base sequence content calculated?
Per Base Sequence Content plots out the proportion of each base position in a file for which each of the four normal DNA bases has been called. In a random library you would expect that there would be little to no difference between the different bases of a sequence run, so the lines in this plot should run parallel with each other.
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.