Lab Matters Summer 2019 - Page 20

FROM THE BENCH South Carolina Automates WGS for Foodborne Outbreak Surveillance By Laura M. Lane, PhD, Molecular Microbiology Lab Supervisor, South Carolina Department of Health and Environmental Control (SC DHEC), Megan Davis, MS, Microbiology Division Director, SC DHEC, Haley Flores, Laboratory Technologist, SC DHEC and R. Brent Dixon, PhD, Public Health Laboratory Director, SC DHEC The common goal of public health laboratories (PHLs) is to provide specialized laboratory testing for the accurate screening, diagnosis, prevention and surveillance of disease, foodborne illness and congenital disorders to improve public health and quality of life. In line with this goal, whole genome sequencing (WGS) has produced genomic information that has been instrumental in identifying congenital disorders, recognizing mutations involved in cancer progression and tracking disease outbreaks. While WGS is often associated with sequencing human genomes, its scalability, applicability to all organisms and ability to produce large volumes of high-resolution data have made it the leading method for performing bacterial foodborne disease surveillance and tracking outbreaks internationally. Compared to pulse-field gel electrophoresis (PFGE), WGS is highly sensitive and data collected is much more detailed. This data can be used for outbreak clustering, to identify molecular commonalities among resistant microbiological strains and to determine early intervention points within food safety. Although WGS brings major advances in data quality and quantity, it still has its challenges. Avoiding sample contamination and preparation errors, improving staff competency training and developing proactive method improvement procedures all contribute to addressing quality control challenges that arise during WGS. This summary highlights how the implementation of automated WGS preparation techniques addresses some of WGS’ most common wet lab challenges. 18 LAB MATTERS Summer 2019 Library Preparation Sequencing clinical samples with the Illumina MiSeq. Photo: SC PHL Samples processed using automated WGS methodologies can be managed in greater numbers (up to 96 samples at a time) with a large decrease in error. Sample Processing High-quality sample DNA input is paramount to successful WGS, as most library preparation protocols depend on accurate input DNA and precise quantifications to obtain optimal fragment sizes, sample coverage, cluster density, etc. Impurities and poor template DNA quality can also be problematic; they negatively affect many enzymatic stages during WGS library preparation. To improve the efficiency of WGS DNA extraction protocols, automated DNA extractors, such as the Qiagen QIAcube, offer high-throughput DNA extraction options and improve sequence concentration and purity. Automation of this WGS step also reduces cross contamination of samples, which subsequently lessens the number of samples that have to be re-sequenced. During WGS library preparation, extracted sample DNA is sheared, either mechanically or enzymatically, and tagged with a universal overhang (i.e., adapters and indices (barcodes)). Samples then undergo several cycles of PCR amplification in preparation for sequencing. During this process, indices are individually added to samples to create unique combinations so that samples can be identified post- sequencing. Errors during manual addition of indices can lead to missing barcodes on samples and/or samples that share the same index combination. This affects downstream analysis of sequencing data as some samples could contain non-index reads while others could be indistinguishable from each other. The Eppendorf epMotion ® 5075 liquid handler provides an automated solution that reduces errors arising from manual index assignment. Prior to sample library preparation, an index plate containing multiple unique index combinations is created by the epMotion ® , and this plate is then used to add unique index combinations to samples in an automated fashion. This ensures that each sample receives a distinctive barcode for sequencing. Tagmentation, which involves transposons cleaving and tagging double- stranded DNA with a universal overhang, determines the success of the library prep, since libraries that are too small (<200bp) or too large (>1.2kb) do not cluster well on the flow cell. A second challenge observed during WGS library preparation involves over- or under-tagmentation of sample DNA. This can occur for a number of reasons (poor initial DNA quality, enzymatic inhibitors, etc.) but, more PublicHealthLabs @APHL APHL.org