NGS for 16S rRNA Microbiome Sequencing

NGS for 16S rRNA Microbiome Sequencing

Norgen Biotek offers comprehensive services for Next-generation sequencing (NGS) using the Illumina Platform in an accredited state-of-the-art laboratory with extensive expertise in sample preparation, sequencing and analysis of all types of environmental and human microbiome samples including soil, water, agricultural products, stool, saliva, blood, etc.

Our expert staff will assist in the initial consultation from sample handling, depth of reads that will be required to answer your questions, the specific variable regions targeted, sample multiplexing per run, to the final analysis of data presented in an easy to understand report. Your project will receive full attention by experts and will guarantee fast turn-around time. Our services include sample isolation, DNA QC, library preparation, sequencing, and analysis of data.

• Comprehensive services - sample-to-answer
• Expertise in isolating and sequencing with environmental and human microbiome samples
• Close consultation and expert advice throughout the process
• Quality assessment of DNA at every step of the process
• Fast turn-around and very responsive staff
• Accredited lab with the highest standard of proficiency

Service Workflow:

1. Consultation - It is essential to have an initial engagement regarding the samples and the project with our experts to define and set clear objectives and outcomes. This is an excellent way to start and it is completely free-of-charge.
2. Sample Submission - We will provide instructions on how to handle, process, and ship the sample and specify the sample requirement (based on sample type). In addition, we will offer sample isolation services.
3. Sample Isolation - We offer tremendous expertise for all sample extractions including low concentration DNA samples, as well as environmental and human microbiome sample types. We offer high quality kits for sample isolation or we perform the sample isolation at our lab with scientists that have expertise in the sample type.
4. DNA Quality Control (QC) - We perform quality control on the isolated DNA to assess quantity, quality, and amplifiability prior to library prep. We share this data prior to proceeding with sequencing.
5. Library Preparation - We generate library preps using industry standard kits for 16S rRNA libraries.
6. Sequencing - We use Illumina platform for sequencing 16S rRNA for profiling and discovery.
7. Analysis - Complete Bioinformatics analysis.
8. Report - A comprehensive report is the final deliverable and is submitted in a timely fashion.

16S NGS Data Analysis Services Available

1. Relative abundance of predominant phyla across samples
2. Proportion of taxonomic assignments under any taxonomic level
3. Relative abundance of bacterial genera across samples
4. Number of phylotypes and genera as function of the total number of sequences
5. Comparison of the microbiotas from different sample origin
6. Group-average clustering of data
7. Detection (identification) of viral, bacteria or fungi sequences

Saliva Samples

Figure 1. Illumina MiSeq 16s rRNA microbiome data from saliva samples preserved for over 6 years using Norgen’s Saliva DNA Collection and Preservation Devices (Cat. RU49000). The saliva DNA was isolated using Norgen’s Saliva DNA Isolation Kit (Cat. RU45400) from saliva that had been preserved for various periods of time up to 6 years at room temperature. A) Principal Coordinate Analysis of 12 saliva microbiomes showing differences in the distribution of taxonomic classifications between samples up to kingdom level. B) Hierarchical clustering of 12 saliva microbiomes based on genus-level classifications, including a bar chart showing the relative abundance of genus-level classifications for each sample in the dendrogram.

Soil Samples

Figure 2. High quality of soil DNA was successfully isolated from the 5 challenging soil types using Norgen’s Soil DNA Isolation Kits (A: Soil DNA Isolation Kit (Magnetic Bead System) and B: Soil DNA Isolation Kit Spin Column) respectively. The Hierarchical Clustering Dendrogram is based on genus-level classifications and shows the relative abundance of its genus-level classifications among soil types.

Soil Samples

Figure 3. Principal Coordinate Analysis (PCoA) generated by Illumina MiSeq. Principal Coordinate Analysis of 20 samples (25-28: Mulch, 29-32: Clay, 33-36: Potting soil, 76-78: Top soil, 79-82: Compost) showing differences in the distribution of taxonomic classifications between samples up to species level. Each soil type is clearly clustered together and away from other types.

Stool Samples

Figure 4. A) Principal Coordinate Analysis of 10 fecal microbiomes showing differences in the distribution of taxonomic classifications between samples up to class level. B) Hierarchical clustering of 10 fecal microbiomes based on genus-level classifications, including a bar chart showing the relative abundance of genus-level classifications for each sample in the dendrogram.

Stool Samples

Figure 5. 16S microbiome data generated by Illumina MiSeq. Stool DNA was isolated using Norgen’s Stool DNA Isolation Kit (Magnetic Bead System) from 200 mg of stool from healthy donors, and the stool microbiomes was successfully sequenced by Illumina MiSeq.

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Norgen Biotek 16S rRNA Microbiome Sequencing Process Workflow: