INFECTIOUS DISEASE | Diagnostic Technology

qPCR + Targeted 16S/ITS NGS Diagnostic Technology

DNA detection with the greatest clinical utility

MicroGenDX combines qPCR with targeted 16S/ITS NGS to deliver timely, highly accurate, actionable diagnostic information for infections. Matching samples against a curated database of over 50,000 microbial species, MicroGenDX’s qPCR+NGS results identify all potentially pathogenic microbial taxa, along with their clinically relevant distribution in the sample, in 3.5 days, at a low cost. In contrast, the strain-level resolution and discovery power of shotgun metagenomics does not provide any greater diagnostic utility for most infections* — and shotgun is also slower and more expensive, with lower sensitivity and accuracy than targeted NGS. For clinical applications, qPCR and targeted NGS offer a clear advantage for optimal treatment strategies.

Comparison of DNA sequencing technologies

	qPCR	Targeted 16S/ITS NGS	Shotgun Metagenomics**
Overview	Amplifies the target gene if found in the specimen Inclusion of specific primers/probes enables detection of those specific microbes	Amplifies and sequences a universal target gene within the microbe genome — not limited by a targeted primer mix like qPCR 16S ribosomal RNA is a highly conserved primer binding site for bacteria and contains species-specific sequences in its hypervariable regions The ITS1 region is a commonly used DNA marker for identifying fungal species in metagenomic samples	Shears the entire genome into random fragments, then sequences the fragments and aligns them back to the entire microbe genome Human host DNA is sequenced at the same time as the microbial DNA, because no target genes are selected as they are in qPCR and NGS
Strengths	High sensitivity Fastest processing speed	High sequencing depth enables high sensitivity High discovery power High mutation resolution High relative processing speed	Base-by-base view of the genome, capturing both large and small variants Provides strain-level information most relevant for viral typing, as well as epidemiology tracing and research
Llimitations	Can only interrogate a limited set of mutations No discovery power beyond the primer set	Cannot detect viruses or parasites No strain level specificity	Longest sequencing and data processing time Most expensive Sequence library is much smaller Lowest overall sensitivity Requires the largest amount of nucleic acid

*Shotgun metagenomics may discover additional resistance genes, but MicroGenDX qPCR detects a total of 17 resistance genes from among all main classes of antimicrobials.

**MicroGenDX is also able to provide shotgun metagenomics offered by its RTL Genomics research division.

References

Mitchell SL, Simner PJ. Next-Generation Sequencing in Clinical Microbiology: Are We There Yet? Clin Lab Med. 2019 Sep;39(3):405-418.doi: 10.1016/j.cll.2019.05.003.
https://www.illumina.com/content/dam/illumina-marketing/documents/gated/10143_Shotgun_Metagenomics_Methods_Guide.pdf

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The clinical importance of targeted NGS’s high sensitivity

High sensitivity enables detection of low-frequency microbes in polymicrobial infections. In Illumina sequencing, high sensitivity is achieved through the number of sequencing reads. For the same region, targeted sequencing achieves 5,000x reads as compared to 75x for shotgun sequencing.

Targeted Sequencing

Shotgun Metagenomics

Curated database of over 50K species of bacteria and fungi

MicroGenDX is one of the world’s largest microbial sample processing facility with over 500,000 samples processed with NGS. The MicroGenDX database contains both public sequences and internally validated novel sequences. For example:

Our database includes entries from known species independently verified by ATCC through culturing, and have gone through our entire NGS workflow.
Our database also contains new sequences from publicly available sources like NCBI, but with three major quality control steps to ensure high-quality source validation.

The rigor that MicroGenDX applies to the quality of its bioinformatics pipeline and microbial database goes above and beyond the current standards of many public DNA/RNA datasets.