High quality of reads and bases
Sequencing single amplicon libraries often yields results of low quality due to the reduced diversity in base composition in the primer regions (figure QIAseq 16S/ITS Panels employ phased primers to increases base diversity and quality scores, A and C). To overcome this issue, QIAseq 16S/ITS Panels use a "phased primer" approach (figure Structure of phased primers used in QIAseq 16S/ITS Panels), which incorporates 0–11 additional bases to the 5’-end of the 16S rRNA or ITS primer. The use of "phased primers" results in a shift in nucleotide balance and increases base diversity, leading to an increase in quality scores (figure QIAseq 16S/ITS Panels employ phased primers to increases base diversity and quality scores, B and D).
Efficient use of flow cell throughput
Because of the "phased primer" approach, libraries produced with QIAseq 16S/ITS Panels are sufficiently complex to eliminate the need for PhiX spike-in, thereby enabling the efficient utilization of flow cell throughput.
Low background noise
Bacterial DNA is present in every corner of our daily lives, resulting in an increased risk of contamination during handling and processing of biological samples. Furthermore, manufacturing and processing of enzymes and reagents can introduce exogenous bacterial DNA to the samples being studied. This contamination background decreases the robustness of bacterial profiling. QIAseq 16S/ITS Panels use low-bioburden reagents, which show very low levels of exogenous bacterial contamination in NTC runs (figure The QIAseq 16S/ITS Panels have very low levels of background contamination due to the use of reagents with low bioburden).
Low DNA input
QIAseq 16S/ITS Panels can be used with a bacterial DNA input ranging from 1 pg to 1 ng, allowing users to profile bacterial communities in samples with low biomass (figure The QIAseq 16S/ITS Panels can be used with as little as 1 pg of input DNA). For profiling fungal communities/species, QIAseq 16S/ITS Panels can detect 0.01 ng of fungal DNA in a background of 1 ng of E. coli DNA.
Each panel can be used to multiplex up to 96 samples on an Illumina MiSeq run using the V3 sequencing chemistry at 2x300. The appropriate index kit is necessary to multiplex the required number of samples.
After sequencing, data is analyzed using QIAGEN Bioinformatics CLC Genomic Workbench and Microbial Genomics Pro Suite Module. A custom workflow, which can be further edited as needed, is available for automating FASTQ file import, sample library demultiplexing, quality controlled filtering and trimming, OUT clustering and secondary bioinformatics analysis. QIAGEN Bioinformatics CLC Genomics Workbench with the Microbial Genomics Pro Suite Module enables researchers to produce standardized reports on sample library quality metrics and organism abundance tables, providing numerous options for downstream interactive analysis of microbiome profiles and reporting on experimental results. The Microbial Genomics Pro Suite Module also includes tools for calculating diversity metrics and for comparing the microbial profiles of different samples. CLC Genomics Workbench produces publication quality figures in formats that are readily useable for researchers to present their results.