Ilumina Low quality bases at first 20 cycles + PHIX problem

Question

[alenew.am]

Good morning, in the last two 16S runs performed (Miseq tool), in the fastqc of the reads a low quality is noted in the first 20 positions or so, which then instead increases again. This drop is always present in the fastqc of R1, but sometimes it is not present in R2.

This phenomenon did not occur in an intermediate run related not to 16S but to whole genome sequencing.

The samples processed in both runs are fecal (a non-fecal sample present in the pool however encountered the same problem), extracted with “Stool DNA Isolation Kits” (Norgen Biotek).

The protocol followed is the Illumina one for the amplification of the V3-V4 region of the 16S (https://support.illumina.com/documen...15044223-b.pdf).

The indices used are DNA/RNA UD Indexes Sets A–B (Catalog nos. 20091654, 20091656), and i have already asked to illumina for the compatibility of this indexes in this protocol.

The parameters set in the Miseq Control Software are 251 + 10 + 10 + 251 (due to the index).

Furthermore, in both runs the % of PHIX was much lower than expected (16% in RUN1 and 11% kin RUN2 instead of 25%), with the additional problem that in RUN2, the % of PHIX aligned between read 1 and read 4 (therefore between R1 and R2, not considering the reads related to the indixes) drops by several points.

Can you tell me a possible reason for these phenomena?

This is the fastqc R1 of a sample

Answer 1

[GenoMax]

Decrease in Q scores manifests itself when the sequence has low nucleotide diversity. Since this is 16S data that likely the case here. You did not include the nucleotide distribution plot but it will likely reflect this. This should not affect the analysis.

% of PHIX was much lower than expected

Since the cluster formation is a stochastic process don't expect to get exactly 25% phiX. phiX is mainly included to mitigate the low-nucleotide diversity (basecalling software has problems when every cluster starts lighting up in field of view, due to identical sequence).

What was the cluster density for these runs? Sometimes deliberate underloading (along with phiX) may need to be employed to avoid these drops.

[alenew.am]

Very thanks for your reply! The cluster density for these runs are 811 and 853; the loading concentration of the DNA is always 8 pM. In the laboratory practice, we finished with 600 uL DNA solution at 8 pM, and replace 120 uL of this solution with 120 uL of PHIX 8 pM, to obtain circa 20-25% PHIX (this is because the PHIX solution at 10 nM over the time slightly increase is concentration). We always check that the initial DNA concentration of the pool is 4 nM (before the process of denaturation and dilution), the size of the fragments is 630 bp circa, and we have also checked the concentration of the PHIX and is correct. In all our other runs 16S this problem with the PHIX has never happen

[GenoMax]

Those cluster densities sound perfect. Whoever is running your sequencers know their stuff. As for why this particular run does not show 25% phiX, it would be hard to pin that down. Could be libraries, how the tech pipetted things that day, phase of the moon, in short difficult to conclude.

You could check with Illumina support to see if they see anything from the run stats but more than likely this would be considered a "acceptable" run.