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?
Attached are the fastqc of R1 of a sample of the first run and a sample of the second run.
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?
Attached are the fastqc of R1 of a sample of the first run and a sample of the second run.