Guidelines for Sanger sequencing and molecular assay monitoring

Abstract

Genetic sequencing, or DNA sequencing, using the Sanger technique has become widely used in the veterinary diagnostic community. This technology plays a role in verification of PCR results and is used to provide the genetic sequence data needed for phylogenetic analysis, epidemiologic studies, and forensic investigations. The Laboratory Technology Committee of the American Association of Veterinary Laboratory Diagnosticians has prepared guidelines for sample preparation, submission to sequencing facilities or instrumentation, quality assessment of nucleic acid sequence data performed, and for generating basic sequencing data and phylogenetic analysis for diagnostic applications. This guidance is aimed at assisting laboratories in providing consistent, high-quality, and reliable sequence data when using Sanger-based genetic sequencing as a component of their laboratory services.

Suggested guidelines for validation of real-time PCR assays in veterinary diagnostic laboratories

“…Should the modified assay result in a more sensitive assay, the analyst could sequence the amplicon to resolve whether the additional detections are specific to the target, as discussed in a companion article in this focus issue and elsewhere. 14,15…”

“…2 In addition to daily monitoring of the assay, primer and probe sequences should be monitored routinely to determine whether emerging/novel nucleotide sequence mutations in the target region alter the performance of the assay. 15 As a final note, tests offered to a client must be completely validated, and the test method should be communicated with the client on the test report. If the client requests use of a non-validated test method (e.g., use on a new species or specimen matrix) and the laboratory conducts such testing, there must be a disclaimer statement added to the test report in order to be compliant with AAVLD accreditation requirements.…”

Time to get real with qPCR controls: The frequency of sample contamination and the informative power of negative controls in environmental (e)DNA studies

“…Bayes' Theorem reminds us to evaluate posterior probabilities in the context of base rates; when the base rate of contamination and the probability of amplification given contamination are low, then there is a considerable likelihood that these very few negative control amplifications were not caused by contaminating substances and were instead other sources of error ("false positive test"; Jerde, 2021). Presence of contaminated DNA in amplified negative controls can be sequence-verified, but this is costly and, as was the case in this study, can result in ambiguous results when DNA targets are short (<150 bp) and amplification occurs at late cycles (Crossley et al, 2020). A rerun of all negative control samples should provide additional insight as to the likelihood of contamination vs. false positives; though, our cumulative probability estimates (p* ) do suggest that there is still a low probability of consistently detecting contaminating DNA (Fig X).…”

Special issue on applied next-generation sequencing in veterinary diagnostic laboratories

“…Use of Sanger sequencing has matured, and it has guidelines for use and monitoring. 4 NGS is currently being used in human and veterinary testing laboratories, with some assays cleared by the FDA for testing of human samples. As the technologies advance and the cost of sequencing has decreased, these types of assays will likely gain further traction in the veterinary diagnostic arena.…”