Fluorescent images (top row) taken from the first cycle of read 1 (R1) showing uneven base distribution in an unphased primer run (A) and more even base distribution in a phased primer run (B). Low base diversity and read quality scores (bottom row) are apparent in a run using unphased V3V4 primers (C) and significantly improved using phased V3V4 primers (D).
Sequences shown may not represent actual primer sequences.
Contamination levels were monitored by amplifying with PCR primers targeting 16S (bacteria) and 18S (fungal) rRNA genes with UCP Multiplex Mastermix and UCP PCR Water. 38 cycles of PCR were performed and the reaction was run on a QIAxcel to detect the 16S and 18S amplicons.
Serial dilutions of a mixed bacterial DNA sample were used to generate libraries using the QIAseq 16S/ITS Region Panel. PCR for 12, 16 or 20 cycles was carried out followed by index PCR. The CLC Microbial Genome Module and SILVA database were used to perform classification.
QIAseq 16S/ITS Panels were used to generate libraries from the ATCC 20 Strain Even Mock Community. Demultiplexing of the variable regions was performed using the CLC Microbial Genomics Workbench and the QIAseq 16S/ITS plugin. Classification was performed for each of the variable regions at the species level using the SILVA database. Results are shown only for Streptococcus mutans. Only a subset of the variable regions (red arrows) can be used to classify S. mutans.