Lab Matters Fall 2024 | Page 100

APHL 2024 POSTER ABSTRACTS

trimming , primer removal and ITS region extraction in bioinformatics pipelines . So , we evaluated workflows with and without fixed length read truncation . We also assessed workflows with and without running the ITSx utility , which was created specifically for ITS region detection . Bioinformatics method-database combinations were evaluated both on degree of classification resolution and computational metrics . Classification resolution was calculated as the F-score for the genus and species levels . Computational burden was assessed based on disk space usage , memory consumption , CPU consumption , time to run and ease of use . This evaluation is an important step in refining laboratory and analytic methods for use in environmental and clinical surveillance and outbreak settings . We plan to conduct similar evaluations for shotgun metagenomic sequencing .

Presenter : Zachary Mudge , xnv5 @ cdc . gov

4.5-79.2 ng /µ L ). The expected size amplicon bands (~ 400 base pairs ) were obtained for RSV A and RSV B samples . Bands were not observed for both NTCs . Approximately 80 % of sequencing reads mapped to the RSV genome for 22 of 22 ( 100 %) RSV A and 16 of 20 ( 80 %) RSV B samples . Genome mean percentage covered at 10X depth was 94 % for RSV A samples and 91.8 % for RSV B samples . All 22 RSV A and 19 of 20 ( 95 %) RSV B samples could be assigned to an RSV Clade .

Conclusions : This tiled amplicon-based approach yields DNA of sufficient quantity and quality for use in whole genome sequencing of RSV A and B . WSLH will use this approach on additional archived RSV-positive specimens to assess RSV mutation rates and diversity in the prevaccine era . These results will allow WSLH to perform prospective RSV genomic surveillance .

Presenter : Matthew Martin , tqe7 @ cdc . gov

Evaluation of a Tiled Amplicon-based Approach for Whole Genome Sequencing of Respiratory Syncytial Virus in Wisconsin , 2023

M . Martin 1 , E . Hanson 2 , C . Jossart 2 , K . Florek 2 , A . Bateman 2 , Centers for Disease Control and Prevention 1 , Wisconsin State Laboratory of

Hygiene 2

Background : Respiratory syncytial virus ( RSV ) is a singlestranded RNA virus that causes mild , cold-like symptoms in most persons . However , infection can be serious in infants and older adults . In the United States , RSV is the leading cause of infant hospitalization and causes ~ 60,000 – 160,000 hospitalizations and ~ 6,000 – 10,000 deaths yearly in adults aged ≥60 years . The Food and Drug Administration recently approved an RSV antibody treatment for infants aged ≤8 months , with an additional dose available for children aged 8 – 19 months at increased risk for severe RSV disease . Two vaccines were approved for adults aged ≥60 years and one vaccine is also approved for use during pregnancy . These advances might cause RSV to mutate and evade these new prevention and treatment tools . The Wisconsin State Laboratory of Hygiene ( WSLH ) has methods to detect RSV but not for sequencing its genome . We evaluated a recently described ( Maloney et al .) tiled amplicon-based method for sequencing RSV . Developing and validating methods to sequence RSV will allow WSLH to enhance RSV surveillance in Wisconsin and contribute to national RSV surveillance .

Methods : Using an EMAG ® automated nucleic acid extraction system , RNA was extracted from historical frozen upper respiratory swab samples that previously tested positive for RSV A ( n = 22 ) or RSV B ( n = 20 ) with the Luminex NxTAG ® respiratory pathogen panel . One negative control ( NTC ) per subtype was also extracted . RNA was reverse transcribed and PCR-amplified using primer pools containing 50 tiled amplicons for RSV A and a separate pool of 50 tiled amplicons for RSV B . After amplification , size of PCR fragments was assessed using a QIAxcel DNA High Resolution Kit and DNA quantity was measured using a Quant-iT dsDNA Assay Kit . DNA was prepared for sequencing using an Illumina DNA Prep kit and sequenced with Illumina NextSeq . Sequencing quality and similarity to available RSV sequences for Clade assignment were assessed using nf-core / viralrecon analysis pipeline .

Results : The concentration of DNA obtained from all samples post amplification was suitable for sequencing ( mean : 52.4 ng /µ L , range :

Evaluation of RSV Genomic Diversity and Variant Prevalence Across Three Respiratory Seasons in Pediatric Cases in Colorado

D . Polanco , A . Alford , D . Mallal , A . Rossheim , L . Bankers , S . Dominguez , S . Matzinger , Colorado Department of Public Health and Environment

Background : Respiratory syncytial virus ( RSV ) is a seasonal respiratory virus that can cause severe disease , particularly in premature infants , young children , immunocompromised people and elderly individuals . Typically , RSV season starts in the fall and peaks during the winter . Annually , RSV infection results in an estimated 58,000-80,000 hospitalizations and 100-300 deaths in children younger than five years of age and 60,000-160,000 hospitalizations and 6,000-10,000 deaths in adults aged 65 years and older . In recent years , RSV has not followed its typical pattern and unlike other respiratory pathogens , such as SARS-CoV-2 and Influenza , there is little genomic surveillance for RSV . Given the annual baseline severity of RSV infection , the effects of SARS-CoV-2- related public health measures on RSV circulation and development of an RSV vaccine for older adults and those who are pregnant , as well as an antibody treatment for very young children , it is important to perform genomic surveillance of RSV to inform both targeted RSV and holistic public health action during the respiratory season .

Methods : We used a duplex RSV A / B PCR assay to screen 650 samples for RSV positivity and A / B subtyping from across the 2021 , 2022 and 2023 ( ongoing ) RSV seasons from patients at Children ’ s Hospital Colorado . We evaluated two amplicon and one tiled amplicon whole genome sequencing ( WGS ) protocols and developed a bioinformatics workflow to analyze RSV WGS data . The workflow was written in workflow description language ( WDL ) and split the process into two phases : assembly and summarization . The assembly phase progresses through tasks for sequence quality assessment and filtering , reference genome alignment , primer trimming , variant and consensus calling , clade assignment using Nextclade and percent genome coverage and depth of coverage calculations . The summarization phase aggregates results from the assembly phase for reporting .

Results : We optimized a WGS method and analyzed results for 650 RSV samples using our bioinformatics workflow . We evaluated the prevalence of RSV A / B subtypes and clades therein , across three respiratory seasons and performed phylogenetic comparisons to