The MicroGenDX NGS Process – Perfected by Experience and Continuous Quality Improvement

Quality nucleic acid extraction

Nucleic acid extraction from multiple specimen types using tailored proprietary techniques that include concentrating cellular material, mechanical lysis, and removing organic inhibitors and human host DNA. Every specimen is processed with a positive and negative extraction control to assure quality.

Illumina MiSeq sequencing process

Illumina is the leading DNA sequencing platform, offering the lowest error rate of < 0.4%. Illumina’s 16S and ITS target gene deep sequencing afford high sensitivity, high discovery power, and high mutation resolution, with three positive and one negative control run alongside every specimen throughout the entire sequencing process to ensure the highest sensitivity and specificity possible.

Superior, curated, bacterial and fungal sequence database

Over 750,000 samples processed using a validated microbial database of over 57,000 referenced microbes, ensuring representation of every possible infective organism, resulting in 14 years of molecular testing quality and reliability.

Sophisticated bioinformatic pipeline

Multi-step denoising and diversity analysis removes sequencing reads errors and chimeras, and applies custom algorithms to provide confident taxonomic assignment.

Nucleic Acid Recovery and Purification Process

Steps in the process

The first step in the process is to extract and purify the DNA from all the clinical samples. Some samples like urine and tissue will require additional preprocessing steps to prepare for extraction. Such steps include centrifugation or slicing specimens into smaller pieces. Then each specimen is mechanically lysed using the bead-beating method on the Qiagen TissueLyser.

Now that the cells have been broken open, the DNA is purified using automated extraction. A positive internal control is added to each specimen at the first stage of extraction to ensure that the extraction process was successful. A negative control follows the entire extraction process in order to identify contamination or background.

A critical element is mechanical lysis, which is used to ensure we are performing the best possible method of extracting bacteria as well as yeast, fungi, and molds from different sample sources. In addition, it is essential to have a successful DNA extraction since all downstream testing is dependent upon high quality DNA.

Illumina MiSeq Sequencing

Work Flow

Sequencing

During sequencing, MicroGenDX runs positive and negative controls through the entire process to view any reagent contamination.  Even though all our reagents and supplies are sterile, the positive controls ensure we properly identify known targets at their known concentration throughout the entire process.  The negative controls ensure that we can identify any known contaminates that could have been introduced during lab procedure steps.  If any contaminates are present, then they are identified and removed during the bioinformatics stage of the NGS process.

A Sophisticated Bioinformatic Pipeline

The MicroGenDX bioinformatic pipeline separates quality reads from procedural noise and performs taxonomic assignment with the greatest accuracy