Plugin error from itsxpress with AVITI data (FASTQ quality value (45) above qmax (41)

Hi @Irshad ! And welcome back to the :qiime2: forum.

Sorry for the delay, I've been thinking how to address this issue properly and time flies!

The error you remark is raised because quality scores in FASTQ files generated from Illumina sequencing should not exceed 41. However, since AVITI was used in this case, it is possible to obtain quality scores as high as Q50!

The solution to the issue appears to be as the error suggests: using the --fastq_qmax 45 option in vsearch (one of the steps within ITSxpress pipeline). AFAIK we cannot modify that option within q2-itsxpress, so maybe the best option here is to adapt your data to Illumina format.

Looking at the q2-itsxpress tutorial, in the PacBio experimental section, they reformat PacBio scoring to Illumina scoring convention using bbmap reformat.sh. This is their example:

If you look at that maxcalledquality in the bbmap reformat.sh GitHub:

maxcalledquality=41     Quality scores capped at this upper bound.

I've never used reformat.sh so I'm not sure how it works. My intuition tells me that using it with maxcalledquality=41 would convert any quality over 41 to 41. We would lose information but that could be an option.

This option also looks interesting:

recalibrate=f           (recal) Recalibrate quality scores.  Must first generate matrices with CalcTrueQuality

But again, I'm not familiar with the tool. This is just in case you want to take the adventure of exploring.

Of course, another option could be just skipping ITSxpress. But I understand this is not what you want.

Sergio

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