16S rDNA Sequencing for Microbe Identification

Sample Handling and Preparation

• Sample reception, logging, and storage under appropriate conditions upon arrival at our lab.
• Bacterial isolates or mixed community samples.

Nucleic Acid Extraction

• Extraction of high-quality microbial DNA from the submitted samples using optimized protocols based on sample type (e.g., pure culture, mixed culture, environmental samples).
• Quantification of the extracted DNA to determine concentration.
• Assessment of DNA quality (e.g., purity, integrity) to ensure suitability for PCR amplification

16S rDNA Gene Amplification

• Targeted amplification of specific variable regions (e.g., V1-V9, V3-V4) of the 16S rDNA gene using PCR with universal or bacteria-specific primers.
• Addition of unique index sequences (barcodes) to each sample's PCR product for multiplex sequencing.
• PCR Product Processing
• Quality control of the amplified fragments using gel electrophoresis to verify size and purity.
• Preparation of the purified PCR products.

Sequencing

Performing the sequencing run on the platform to generate raw sequence reads.

Bioinformatics Analysis

• Sequence Alignment: Aligning the processed reads against reference databases of 16S rDNA sequences (e.g., NCBI).
• Taxonomic Assignment: Identifying the closest matching sequences in the database to assign a taxonomic classification (e.g., genus, species) to the query sequences.

Data Reporting (Deliverables)

Turnaround Time

• For pure microbial identification using 16S rDNA sequencing, you can generally expect a relatively quick turnaround.
• For the microbes in the mixed sample, it depends on the complexity of the sample.