Report: ZymoBIOMICS® Targeted Sequencing Service for Microbiome Analysis
Zymo Research Corporation
1 Results
Click on the links below to view the results from each analysis:
2 QC Data
The ZymoBIOMICS® Targeted Sequencing Service for Microbiome Analysis includes both negative and positive quality control samples for every project. Negative controls are included to assess the level of bioburden carried by the wet-lab process. Positive controls utilize a mock microbial community of well-defined composition to ensure that the data generated is representative of the analyzed microbial samples. The barplot below shows the microbial composition of the ZymoBIOMICS® Microbial Community Standard (Zymo Research, Irvine, CA) measured in this project. The theoretical composition of the standard can be accessed by clicking on the link below the figure.
For projects that included DNA purification from raw samples, the ZymoBIOMICS® Microbial Community Standard was used as positive control; a blank extraction sample was used as a negative control. For projects that did not include DNA purification (i.e., analysis of DNA samples), the ZymoBIOMICS® Microbial Community DNA Standard (Zymo Research, Irvine, CA) was used as positive control; a blank library preparation sample was used as a negative control.
3 Materials and Methods
The samples were processed and analyzed with the ZymoBIOMICS® Targeted Sequencing Service for Microbiome Analysis (Zymo Research, Irvine, CA).
DNA Extraction: One of three different DNA extraction kits was used depending on the sample type and sample volume. In most cases, the ZymoBIOMICS®-96 MagBead DNA Kit (Zymo Research, Irvine, CA) was used to extract DNA using an automated platform. In some cases, ZymoBIOMICS® DNA Miniprep Kit (Zymo Research, Irvine, CA) was used. For low biomass samples, such as skin swabs, the ZymoBIOMICS® DNA Microprep Kit (Zymo Research, Irvine, CA) was used as it permits for a lower elution volume, resulting in more concentrated DNA samples.
Targeted Library Preparation: Bacterial 16S ribosomal RNA gene targeted sequencing was performed using the Quick-16S™ NGS Library Prep Kit (Zymo Research, Irvine, CA). In most cases, the bacterial 16S primers amplified the V3-V4 region of the 16S rRNA gene. These primers have been custom-designed by Zymo Research to provide the best coverage of the 16S gene while maintaining high sensitivity. Fungal ITS gene targeted sequencing was performed using the Quick-16S™ NGS Library Prep Kit with custom ITS2 primers substituted for 16S primers.
The sequencing library was prepared using an innovative library preparation process in which PCR reactions were performed in real-time PCR machines to control cycles and therefore limit PCR chimera formation. The final PCR products were quantified with qPCR fluorescence readings and pooled together based on equal molarity. The final pooled library was cleaned with the Select-a-Size DNA Clean & Concentrator™ (Zymo Research, Irvine, CA), then quantified with TapeStation®(Agilent Technologies, Santa Clara, CA) and Qubit® (Thermo Fisher Scientific, Waltham, WA).
Control Samples: The ZymoBIOMICS® Microbial Community Standard (Zymo Research, Irvine, CA) was used as a positive control for each DNA extraction, if performed. The ZymoBIOMICS® Microbial Community DNA Standard (Zymo Research, Irvine, CA) was used as a positive control for each targeted library preparation. Negative controls (i.e. blank extraction control, blank library preparation control) were included to assess the level of bioburden carried by the wet-lab process.
Sequencing: The final library was sequenced on Illumina® MiSeq™ with a v3 reagent kit (600 cycles). The sequencing was performed with 10% PhiX spike-in.
Bioinformatics Analysis: Unique amplicon sequences variants were inferred from raw reads using the DADA2 pipeline (Callahan et al, 2016). Chimeric sequences were also removed with the DADA2 pipeline. Taxonomy assignment was performed using Uclust from Qiime v.1.9.1 with the Zymo Research Database, a 16S database that is internally designed and curated, as reference. Composition visualization, alpha-diversity, and beta-diversity analyses were performed with Qiime v.1.9.1 (Caporaso et al., 2010). If applicable, taxonomy that have significant abundance among different groups were identified by LEfSe (Segata et al., 2011) using default settings. Other analyses such as heatmaps, Taxa2ASV Deomposer, and PCoA plots were performed with internal scripts.
4 References
Callahan B.J., McMurdie P.J., Rosen M.J., Han A.W., Johnson A.J., Holmes S.P., (2016) DADA2: High resolution sample inference from Illumina amplicon data. Nat Methods 13(7):581-3.
Caporaso, J.G., Kuczynski, J., Stombaugh, J., Bittinger, K., Bushman, F.D., Costello, E.K. et al. (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7: 335-336.
Segata, N., Izard, J., Waldron, L., Gevers, D., Miropolsky, L., Garrett, W.S., and Huttenhower, C. (2011) Metagenomic biomarker discovery and explanation. Genome Biol 12: R60.
