Lab Matters Fall 2024 | Page 95

APHL 2024 POSTER ABSTRACTS design of a real-time dashboard , this project demonstrates a path forward for comprehensive integration of WGS data into public health workflows .

Presenter : Catharine Prussing , catharine . prussing @ health . ny . gov

Compact Sample Prep for High Containment Molecular Workflows

J . Chami , P . Gilliam , B . Mason , J . Hlavay , Redbud Labs , Inc .

Molecular analysis ( qPCR and sequencing ) are essential tools for public health labs tasked with tracking incidence and genomic variance of SARS , influenza , tuberculosis , fungal infections , biothreats and foodborne illness . Often , these workflows are performed in high containment environments , such as a biosafetylevel 3 ( BSL-3 ) lab , or within a BSL-2 lab where work is performed in a biosafety cabinet ( BSL-2 +). However , most molecular analysis equipment is too large and requires maintenance that makes it incompatible with these environments . As a result , much genomic epidemiology is still performed manually , with the attendant issues for training , user error , cost and consistency .

In an effort to streamline genomic epidemiology for public health labs , we have developed a new compact sample prep platform called NAxtract . We have successfully extracted nucleic acids from a range of pathogens ( e . g . viruses , gram - and gram + bacteria ) in a variety of sample matrices . Here , we show agreement with existing manual and automated viral and bacterial extractions . We report results using a range of analytical methods , including qPCR and sequencing . The NAxtract instrument is approximately one-cubic foot and takes an end user less than five minutes to unbox and set up , with no service support or training required . We discuss the impact of NAxtract on a variety of workflows in terms of hands-on time and overall performance .

Presenter : Jenine Chami , chami @ redbudlabs . com

Comparison of 96 ESBL Strains Whole Genome Sequenced with Illumina and PacBio Technology

J . Monk 1 , G . Young 2 , P . Sankey 3 , C . Wong 3 , A . Souppe 2 , K . Scott 2 , A . Galicia 4 , M . Zahn 5 , Palmona Pathogenomics 1 , Pacific Biosciences 2 , Avellino Lab 3 , Orange County Public Health Lab 4 , Orange County Health Care Agency 5

Overview : A set of 96 ESKAPEE strains possessing extended spectrum beta lactamase ( ESBL ) genes were collected from a local public health laboratory from 2017-2023 . The strains were processed through whole-genome sequencing ( WGS ) using both Illumina short-read and PacBio long-read sequencing technologies . The resulting data examined the differences between the PacBio and Illumina sequencing data generated . These are presented by genome assembly statistics , antimicrobial resistance gene identification , virulence factor mapping and epidemiology applications .

Results : Genome assemblies resulting from PacBio sequencing were more complete than those generated from Illumina assemblies . The average coverage for Illumina sequencing was 263x +/ -51 with a minimum coverage of 134x and maximum coverage of 388x . while the average coverage for PacBio sequencing was 182x +/ - 58 with a minimum coverage of 56x and maximum coverage of 364x . The average number of contigs per genome for assemblies resulting from Illumina assembles was 167 +/ -57 while the average number of contigs from PacBio assemblies was 3 +/ - 1 ( including both chromosome and plasmid sequences ). The average N50 score for Illumina assemblies was 1.99 Mbp while the average N50 score for PacBio assemblies was 4.99Mbp , close to the expected size of an ESKAPEE full genome . PacBio sequencing resolved 92 out of 96 ( 96 %) full chromosome sequences for the strains of which 82 ( 89 %) were fully circularized . Another 153 potential plasmids were completely resolved from the PacBio sequencing runs with an average of 1.6 +/ - 0.9 plasmids detected per strain . 35 of these plasmids are mapped to known plasmids in the PLSDB database . Palmona ’ s Pathogenomics Platform ( P3 ) was used to evaluate differences in AMR gene identifications and VF mapping observed between the two sequencing methods .

Methods : For Illumina sequencing , libraries were prepared using the seqWill plexWell 96 kit protocol . Samples were normalized to 12.5 ng input using the Tecan Freedom Evo prior to sample barcoding , plate barcoding , library amplification and purification steps . Pooled libraries were diluted to a final concentration of 2nM and loaded onto an Illumina NextSeq1000 using a NextSeq 1000 / 2000 P2 reagent kit ( 300 Cycles ). DNA sample and library QC was done using the Varioskan LUX and Agilent Tapestation 4200 System . For PacBio sequencing , bacterial DNA was mechanically fragmented using the Hamilton NGS STAR system and libraries prepared using the new HiFi prep kit 96 protocol without any sample input normalization . Input amounts ranged from 120 to 1350 ng and all libraries were pooled after the indexed adapter ligation step . Sequencing polymerase was bound to the 96-plex pool and loaded on the Revio system at a concentration of 200 pM . The 96-plex pool was sequenced on a single Revio SMRT Cell using v13 software and 24-hour sequencing movie time . Read QC / QA was conducted with FastQC v0.11.9 for both platforms . Illumina genomes were assembled using Unicycler v0.4.8 with Spades v3.13 . PacBio genomes were assembled with Flye v2.9.3 . Genome assemblies were evaluated using Quast v5.2.0 . All genomes were annotated with Prokka v 1.14.6 . AMR genes were called using AMRfinder v 1.0.13 and virulence factors were identified using Abritamr v1.0.1 .

Presenter : Jonathan Monk , jonathan . monk @ palmona . com

Detecting Nosocomial Spread of Candida auris by Straintyping with Whole Genome Sequencing

S . Murphy 1 , E . Keller 2 , T . Ross 1 , A . Fitzgerald 1 , T . Moore 2 , W . Memon 1 , E . Barker 1 , M . Curless 1 , P . Simner 1 , S . Zhang 1 , Johns Hopkins Medicine 1 , Maryland Department of Health 2

Background : Candida auris , an emerging multidrug resistant fungal pathogen , poses a significant challenge due to its propensity for nosocomial spread and potential to cause invasive infections . The ability to identify closely related C . auris strains is a critical component of epidemiological investigations and infection prevention . Since 2022 , an uptick in C . auris cases was observed in multiple hospitals within our healthcare system . In this study , we utilized whole genome sequencing ( WGS ) to assess genetic relatedness of over 40 C . auris strains ( 26 cases ) to identify potential nosocomial transmission .

Methods : DNA was extracted using manual methods ( Zymo ) and WGS was performed using Illumina-based platforms ( MiSeq and NextSeq ). Data was analyzed , in collaboration with the Maryland Department of Health , using an open source bioinformatic pipeline