I am confused as to how quality scores are actually calculated by DNA sequencers like Illumina. For each base call, some quality predictor value is computed, based on various properties of the sequencing machine, like intensity of light during the read.

Do we know exactly how these quality scores are computed? Exactly how many factors go into computing these QUAL values?

  • $\begingroup$ only have experience with Illumina platforms here's a link to the basics Illumina quality info More detailed Illumina info Take a look at the refs in that document to go deeper. (Am assuming you don't mean variant call quality scores which, in the pipeline I use, is all handled post sequencing) $\endgroup$ – user3234810 Apr 10 '16 at 2:27
  • $\begingroup$ @user3234810 Thanks. But I was looking for more information than that. There's some relationship between "signal-to-noise" and quality scores. How exactly Illumina calculates these, I don't know. $\endgroup$ – ShanZhengYang Apr 10 '16 at 14:27
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    $\begingroup$ The quality score calculations are different for different types of machines. Specifically interested in Illumina? $\endgroup$ – WYSIWYG Apr 11 '16 at 5:37
  • $\begingroup$ @WYSIWYG Actually, any sequencer would do. I just mentioned Illumina as a starting point. $\endgroup$ – ShanZhengYang Apr 11 '16 at 14:33
  • $\begingroup$ @ShanZhengYang I would suggest that you restrict your question to one kind of machine (or kinds of machine with same base calling technique). You can stick to just Illumina. Moreover, even for illumina there are different base calling approaches. $\endgroup$ – WYSIWYG Apr 11 '16 at 14:55

I'm restricting this answer to Illumina. Even then, I don't know about the exact details of the raw data analysis (it is a proprietary software).

Basically Illumina records the sequence based on photographic images. Each nucleotide has a distinct fluorescent label. In a cycle, a nucleotide is pumped and unincorporated nucleotides are washed off (this is repeated for all nucleotides). A laser excites the fluorophore and the emitted light is recorded in the form of a photograph. The template DNA is present in the form of clusters of strands (at a given location), which enables easy visual identification of the fluorescence.

Base calling is done using image analysis. Each image is analysed for intensities of different colours and based on this the quality score is calculated. The quality score is basically the log likelihood of a occurrence nucleotide at a given position (based on its colour intensity) compared to other nucleotides.

This is the most simple explanation of how Illumina does base calling. There are different kinds of errors and biases and there are different statistical approaches to correct for them.

Have a look at following references for more details:


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