QIIME 2 processing of MiSeq i100 data with constant quality scores

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Hi all,

Apologies for raising a question that has already been discussed in previous threads, but I am still unsure how to proceed with my dataset.

Related posts:

I have recently received amplicon sequencing data generated on the Illumina MiSeq i100 platform (16S rRNA gene, 515F/806R primer pair). When importing and visualising the demultiplexed reads in QIIME 2, the interactive quality plots show high and constant quality scores at Q38.

Interactive quality plots
Interactive quality plots1545×465 16.1 KB

Given this unusual lack of quality variation and decay, I am unsure how appropriate it is to proceed with the standard DADA2 denoising workflow in QIIME 2.

In particular, I have two main questions:

  1. Is it valid to proceed with DADA2 denoising on MiSeq i100 data in the usual way?

  2. Without the typical quality score drop-off, what is the recommended approach for determining truncation parameters?

Any guidance or shared experience would be greatly appreciated.

Thank you very much,

Kat

2

Hello @Kat,

  1. The developer of DADA2 made a post about the compatibility of the software with binned quality scores here.

  2. It might simply be the case that no truncation is needed, if truncation were only going to be applied to remove low quality bases.