Commentary: Call for a Quality Standard for Sequence-Based Assays in Clinical Microbiology: Necessity for Quality Assessment of Sequences Used in Microbial Identification and Typing

Abstract

Subsequent to the early days of radioisotope sequencing, DNA sequencing technologies have evolved rapidly, giving rise to techniques that are more sensitive and rapid and provide longer sequence reads. These incremental improvements in technology and in the automation of modern sequencing platforms have resulted in applications of DNA sequencing across virtually all biological science disciplines. In microbiology, this has led to a significant increase in the number of available complete microbial and viral genome sequences and, consequently, to increasing use of DNA sequence analysis in clinical microbiology laboratories, where the results may be influential on consequent patient management. The evolution of Sangerbased technologies is now being complemented by the revolution of second- and third-generation sequencing technologies that, potentially, could provide whole-microbial-genome analysis at the clinical bench and have a greater, more direct impact on patient and outbreak management. While the technologies have continued to evolve, common standards for the description of DNA sequence data quality still do not exist. Further, utilization of DNA sequence data requires comparison with other similar sequence data held in private or public databases. Similarly, there is no process for the accreditation of these databases and the analyses that they provide to ensure that they are fit for purpose. We strongly urge that action is required to address this. CURRENT USES OF SEQUENCING IN CLINICAL MICROBIOLOGY DNA sequencing is being introduced into microbial diagnosis particularly for the identification of esoteric organisms where traditional methods fail or where the delay to definitive identification by traditional means is significant to patient treatment. The most common locus used for bacterial identification is the 16S rRNA gene (6). In general, the complete sequence data rather than individual bases are important when making comparisons to existing data to make the identification. Other loci, in particular rpoB and gyrB (19, 31), are often used for genera whose species are more difficult to resolve with the 16S locus. Because the identification may be used in clinical management to alter treatment regimens, the quality of the data used to make this identification is crucial. Sequence-based typing methodologies, typified by multilocus sequencing typing (MLST), are often used for typing in epidemiological studies and population biology. The results do not usually have a clinical impact in the short term but may be of great importance in medico-legal cases when identifying source of infection, and therefore data quality is essential. Crucially, with these techniques, a single-base change alters the type; therefore, the quality at individual bases is as relevant as