Bizzare quality plots

[Laura_Scannell]

Hello everyone, I'm relatively new to Qiime2 and bioinformatics in general. I've been working on a project with a very small sample size and low predicted species count. This is my Quality score graph after sequencing (v4):

I was wondering if this type of high quality scores in weird blocks could indicate anything about problems with my data, could this be from too much primer noise or is this normal for very small sample sizes?

Thank you!

[Nicholas_Bokulich]

Welcome to the forum, @Laura_Scannell!

This sounds like a familiar issue — are you using NovaSeq or iSeq data by any chance? See here for some related discussion:

Odd display of demux plot

I'm pushing some data through the qiime2 pipeline and am met with an odd demux plot, one that makes it difficult to choose parameters for the dada2 step. I am missing most of the box part of the boxplot that every other demux.qza that I've seen has. The plot is generated using random sampling of 1000000 out of 19442585 sequences, and looks exactly the same as when I use 10000 sequences. I wonder if it might be due to Illumina's quality score binning? Please see the demux.qza object below. demu…

@Laura_Scannell

I was wondering if this type of high quality scores in weird blocks could indicate anything about problems with my data, could this be from too much primer noise or is this normal for very small sample sizes?

Some newer Illumina sequencers bin quality scores into 4 bins to reduce the data footprint. See the topic above and here for more details.

Good luck!

[Laura_Scannell]

Thanks a ton, I was able to simply open it in FASTQC and get less blocky plots.

[thermokarst]

Hi @Laura_Scannell - One thing I'll point out about FastQC is that the x axis on those plots it generates can be a little confusing if you're directly comparing to q2-demux's summarize visualization, because FastQC actually performs a separate binning of the nt positions:

• FastQC compared to QIIME 2 quality plot
• Quality in reads suddenly decreases with no apparent reason - #5 by thermokarst

So in particular, you're probably looking at a psuedo-log type of plot, where only ~50 or so box plots are displayed, rather than the ~290 box plots that you shared in the screenshot above.

Just something worth keeping in mind!

[Laura_Scannell]

Thanks a lot, I’m relatively new to everything so I’m glad you brought this up.