Setting DADA2 parameters for ITS reads (with lower quality)

Hi!

I’ve done ITS2 sequencing (MiSeq 2x300 bp V3 ) and my target size should be around 400-500 bp. I’m having a hard time deciding the parameters for DADA2 denonising and specially trunc-len parameter. The problem is that my data quality does not look so good and I’m not sure how to handle this kind of data.

DADA2 will take care of the data by correcting possible errors but I’m concerned about the data quality dropping at the ends of the reads. In bacterial data 16s for example it is okay to use trunc-len to discard reads that start to be bad quality reads. But in ITS it is not recommended to use that because of the natural variability.

What parameters should I use to be sure that the bad quality reads are discarded. Or is this data quality so poor that I should not use this data-set?

Thank you for advance!

Best
Veera

1 Like

Hello Veera,

Sorry to hear about your sequencing run. If at all possible, I would try to sequence this library again and hope for better quality near the ends of your reads, but not all hope is lost!

When using the default dada2 parameters, reads that cannot be joined are discarded, so this will happen just by running dada2!

I would start by running dada2, seeing how many reads are retained and if you can use that as a starting point. If dada2 paired ends has trouble, running dada2 single end on just your forward read could be another good option. You can easily truncate your forward read to, say, 200 bp and see if the higher quality start produces different / better results.

Let me know what you find!
Colin