Implementing laboratory automation for next-generation sequencing: benefits and challenges for library preparation.

In the wake of COVID-19, the importance of next-generation sequencing (NGS) for diagnostic testing and surveillance-based screening has never been more evident. Considering this, continued investment is critical to ensure more public health laboratories can adopt these advanced molecular technologies. However, many facilities may face potential barriers such as limited staff available to routinely prepare, test, and analyze samples, lack of expertise or experience in sequencing, difficulties in assay standardization, and an inability to handle throughput within expected turnaround times. Workflow automation provides an opportunity to overcome many of these challenges. By identifying these types of sustainable solutions, laboratories can begin to utilize more advanced molecular-based approaches for routine testing. Nevertheless, the introduction of automation, while valuable, does not come without its own challenges. This perspective article aims to highlight the benefits and difficulties of implementing laboratory automation used for sequencing. We discuss strategies for implementation, including things to consider when selecting instrumentation, how to approach validations, staff training, and troubleshooting.

Phylogeny and Genomic Characterization of Clinical Salmonella enterica Serovar Newport Collected in Tennessee.

Salmonella enterica subsp. enterica serovar Newport (S. Newport) is a clinically and epidemiologically significant serovar in the United States. It is the second most prevalent clinically isolated Salmonella serovar in the United States, and it can contaminate a wide variety of food products. In this study, we evaluated the population structure of S. Newport clinical isolates obtained by the Tennessee Department of Health during routine surveillance (n = 346), along with a diverse set of other global clinical isolates obtained from EnteroBase (n = 271). Most of these clinical isolates belonged to established lineages II and III. Additionally, we performed lineage-specific phylogenetic analyses and were able to identify 18 potential epidemiological clusters among the isolates from Tennessee, which represented a greater proportion of Tennessee isolates belonging to putative epidemiological clusters than the proportion of isolates of this serovar that are outbreak related. IMPORTANCE This study provides insight on the genomic diversity of one of the Salmonella serovars that most frequently cause human illness. Specifically, we explored the diversity of human clinical isolates from a localized region (Tennessee) and compared this level of diversity with the global context. Additionally, we showed that a greater proportion of isolates were associated with potential epidemiological clusters (based on genomic relatedness) than historical estimates. We also identified that one potential cluster was predicted to be multidrug resistant. Taken together, these findings provide insight on Salmonella enterica serovar Newport that can impact public health surveillance and responses and serve as a foundational context for the Salmonella research community.

Phylogenetic Analysis Reveals Source Attribution Patterns for Campylobacter spp. in Tennessee and Pennsylvania.

Campylobacteriosis is the most common bacterial foodborne illness in the United States and is frequently associated with foods of animal origin. The goals of this study were to compare clinical and non-clinical Campylobacter populations from Tennessee (TN) and Pennsylvania (PA), use phylogenetic relatedness to assess source attribution patterns, and identify potential outbreak clusters. Campylobacter isolates studied (n = 3080) included TN clinical isolates collected and sequenced for routine surveillance, PA clinical isolates collected from patients at the University of Pennsylvania Health System facilities, and non-clinical isolates from both states for which sequencing reads were available on NCBI. Phylogenetic analyses were conducted to categorize isolates into species groups and determine the population structure of each species. Most isolates were C. jejuni (n = 2132, 69.2%) and C. coli (n = 921, 29.9%), while the remaining were C. lari (0.4%), C. upsaliensis (0.3%), and C. fetus (0.1%). The C. jejuni group consisted of three clades; most non-clinical isolates were of poultry (62.7%) or cattle (35.8%) origin, and 59.7 and 16.5% of clinical isolates were in subclades associated with poultry or cattle, respectively. The C. coli isolates grouped into two clades; most non-clinical isolates were from poultry (61.2%) or swine (29.0%) sources, and 74.5, 9.2, and 6.1% of clinical isolates were in subclades associated with poultry, cattle, or swine, respectively. Based on genomic similarity, we identified 42 C. jejuni and one C. coli potential outbreak clusters. The C. jejuni clusters contained 188 clinical isolates, 19.6% of the total C. jejuni clinical isolates, suggesting that a larger proportion of campylobacteriosis may be associated with outbreaks than previously determined.

Genomic characterization and phylogenetic analysis of Salmonella enterica serovar Javiana.

Salmonella enterica serovar Javiana is the fourth most reported serovar of laboratory-confirmed human Salmonella infections in the U.S. and in Tennessee (TN). Although Salmonella ser. Javiana is a common cause of human infection, the majority of cases are sporadic in nature rather than outbreak-associated. To better understand Salmonella ser. Javiana microbial population structure in TN, we completed a phylogenetic analysis of 111 Salmonella ser. Javiana clinical isolates from TN collected from Jan. 2017 to Oct. 2018. We identified mobile genetic elements and genes known to confer antibiotic resistance present in the isolates, and performed a pan-genome-wide association study (pan-GWAS) to compare gene content between clades identified in this study. The population structure of TN Salmonella ser. Javiana clinical isolates consisted of three genetic clades: TN clade I (n = 54), TN clade II (n = 4), and TN clade III (n = 48). Using a 5, 10, and 25 hqSNP distance threshold for cluster identification, nine, 12, and 10 potential epidemiologically-relevant clusters were identified, respectively. The majority of genes that were found to be over-represented in specific clades were located in mobile genetic element (MGE) regions, including genes encoding integrases and phage structures (91.5%). Additionally, a large portion of the over-represented genes from TN clade II (44.9%) were located on an 87.5 kb plasmid containing genes encoding a toxin/antitoxin system (ccdAB). Additionally, we completed phylogenetic analyses of global Salmonella ser. Javiana datasets to gain a broader insight into the population structure of this serovar. We found that the global phylogeny consisted of three major clades (one of which all of the TN isolates belonged to) and two cgMLST eBurstGroups (ceBGs) and that the branch length between the two Salmonella ser. Javiana ceBGs (1,423 allelic differences) was comparable to those from other serovars that have been reported as polyphyletic (929-2,850 allelic differences). This study demonstrates the population structure of TN and global Salmonella ser. Javiana isolates, a clinically important Salmonella serovar and can provide guidance for phylogenetic cluster analyses for public health surveillance and response.