Lab Matters Fall 2023 | Page 74

APHL 2023 POSTER ABSTRACTS

Maximizing MAGs from Long-read Metagenomic Assemblies : A Post-assembly Pipeline with Completenessaware Binning

D . Portik , J . Wilkinson ; Pacific Biosciences

The use of metagenome-assembled genomes ( MAGs ) in microbial surveillance has increased in popularity . Metagenome assembly can allow for fine-scale resolution of epidemiological tracing and improve recovery of antimicrobial resistance ( AMR ) genes . Highly accurate long reads provide major advantages for metagenome assembly . PacBio HiFi sequencing of metagenomic samples with the Sequel IIe system regularly produces reads 8 – 15kb in size with a median QV ranging from Q30 – 40 ( 99.9 – 99.99 % accuracy ). With the development of new metagenome assembly algorithms specific to HiFi reads ( including hifiasm-meta ), it is now possible to reconstruct full metagenome-assembled genomes ( MAGs ) for many high abundance species . However , discontiguous assemblies routinely occur for lower abundance taxa . Post-assembly tools incorporating binning methods are therefore required to identify these MAGs from the assembled contigs . Here , we present the newest version of HiFi-MAG-Pipeline , a comprehensive workflow that automates major steps including binning , quality filtering , and taxonomic identification . The outputs include high-quality MAG sequences , associated metadata , and visualizations of key MAG characteristics . Importantly , the pipeline uses a custom binning strategy that is tailored to long-read assemblies . To demonstrate the performance of this pipeline , we assembled and analyzed several publicly available HiFi metagenomics datasets . We show that complete , circular MAGs are routinely produced from HiFi metagenome assembly , and that HiFi-MAG-Pipeline recovers more total high-quality MAGs than other standard methods . Overall , our results suggest that HiFi sequencing is an effective strategy for obtaining large numbers of high-quality MAGs , which can be used to improve microbial surveillance efforts .

Presenter : Trang Dahlen , tdahlen @ pacificbiosciences . com

Monitoring COVID-19 Variants in Wisconsin Using SARS- CoV-2 Whole-Genome Sequencing in Wastewater

A . Roguet , K . Janssen , D . Antkiewicz , R . Fahney , P . Mullen , H . Pilch , G . Knuth , D . Everett , J . Hemming , M . Shafer ; Wisconsin State Laboratory of Hygiene

SARS-CoV-2 , the viral agent of COVID-19 , poses challenges to the clinical surveillance systems due to large numbers of asymptomatic and therefore untested individuals , the increased use of athome tests , and the limited access to testing facilities for some populations and regions . In Wisconsin , these challenges also affect the number of clinical specimens sequenced , which can lead to a skewed picture of the COVID-19 variant situation , which impedes the ability of the public health community to take appropriate action . Wastewater-based epidemiology ( WBE ) overcomes many of these challenges by testing integrative wastewater samples representative of entire communities served by municipal wastewater treatment facilities . Since 2020 , WBE has demonstrated its utility to serve as a complementary and independent epidemiological tool to assess COVID-19 prevalence and help prevent the spread of COVID-19 in communities . While the monitoring of SARS-CoV-2 concentrations has been popularized within public health laboratories , the use of genomic sequencing of wastewater to monitor COVID-19 variants is far from becoming mainstream . The Wisconsin State

Laboratory of Hygiene ( WSLH ) initiated in January 2022 a statewide wastewater SARS-CoV-2 genomic surveillance program to monitor the prevalence of COVID-19 variants in the population using a tiled amplicon whole-genome sequencing strategy . We demonstrated that wastewater-based prevalence variant data mirrored the successive Omicron waves documented in clinical samples . Thus , it could serve as an early warning system . To make these data accessible to larger audiences , we developed interactive visuals for a publicfacing dashboard . We also developed comprehensive and detailed data visualization tools for internal use to assess the quality of sequencing data , identify emerging mutations , and detect potential cryptic variants . Our bioinformatics workflows rely on free and accessible databases and software and are all publicly available to help other public health laboratories interpret their next-generation sequencing data .

Presenter : Adelaide Roguet , adelaide . roguet @ slh . wisc . edu

Multi-State Evaluation of LRN Rapid BSL-3 Bacillus anthracis Whole Genome Sequencing for Antimicrobial Resistance Markers and Bioengineering Detection for Anthrax Preparedness

B . Cherney 1 , L . Medina-Cordoba 1 , R . Schuester 2 , J . Huynh 2 , D . Centurioni 3 , E . Swaney 4 , D . Hairfield 5 , V . Chivukula 6 , W . Overholt 6 , R . Rahmat 7 , T . Wolford 7 , J . Bugrysheva 1 , P . Michel 1 , T . Maxson 1 , D . Sue 1 ; 1 US Centers for Disease Control and Prevention , 2 Arizona Department of Health Services . 3 New York State Department of Health , Wadsworth Center , 4 Texas Department of State Health Services , 5 Virginia Division of Consolidated Laboratory Services ,

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US Centers for Disease Control and Prevention , Laboratory Preparedness and Response Branch , Applied Bioinformatics Laboratory , 7 Association of Public Health Laboratories

During a public health emergency involving Bacillus anthracis , detection of antimicrobial resistance ( AMR ) will inform decisions about anthrax treatment and post-exposure prophylaxis to save lives . B . anthracis is a bacterium subject to select agent requirements ( 42 CFR Part 73 ). CDC ’ s BioDefense Research and Development laboratory completed a multi-state pilot study evaluation of same-day whole genome sequencing ( WGS ) for B . anthracis in collaboration with the Association of Public Health Laboratories and Laboratory Response Network ( LRN ) laboratories , including Arizona Department of Health Services ( Phoenix , AZ ), Wadsworth Center , New York State Department of Health ( Albany , NY ), Texas Department of State Health Services ( Austin , TX ) and the Division of Consolidated Services ( Richmond , VA ). LRN scientists conducted rapid BSL-3 WGS , with Oxford Nanopore MinION Mk1B sequencers using genomic DNA from agar cultured B . anthracis . Nucleic acid was prepared from culture isolates and sequenced in the same biological safety cabinet , thereby minimizing the time to results and reducing laboratory biosafety risks . Each laboratory completed at least four single-plex and one multiplex sequencing runs . Genomic data sets were analyzed on-site using custom , open-source bioinformatics software called PiMA — a bacterial genome analysis pipeline . The software was installed on the same laptop that powered the BSL-3 sequencer and performed quality control analysis , de novo sequence assembly and identification and characterization of AMR features , including the detection of plasmids , integration sequences and mutations . Both single-plex