How PacBio quality is calculated?

Question

[Medhat]

As the question declares I want to know how pacbio quality is calculated?

If I am using the SMRT portal for example RS_Subreads.1

In the filtering process It will give me this values

SFilter v1
Minimum Subread Length  50
Minimum Polymerase Read Quality  75 
Minimum Polymerase Read Length  50

I can change It but how it is calculated?

I know that there is two basic steps the Basic analysis step which begins with movie -> puls -> kinetic measures -> and at last bases

the second step which is called secondary analysis, using the smrt portal which I can change the numbers in each algorithm but how it is related to the main sequences?

Thanks,

Answer 1

[rhall]

In a polymerase (raw) read each base has a set of QVs based on the probability of that base being incorrectly called when calculating base calls from pulses. These QVs include probability of substitution, deletion, insertion and pulse merging. The polymerase read quality is calculated as an estimation of error from the set of QV values for each base in the read. The read quality estimation is conservative, you can see this by comparing the RQ distribution to the mapped accuracy distribution for a known sample.

[philippe.leroy.2]

The definition is great. But it doesn't explain clearly how to calculate this QVs using a bam file obtained by mapping reads against the consensus using balsr and/or hisat2 for example?

Are there a list of tools to use to do such a calculation

Thank you in advance

Best
Philippe