Lab Matters Summer 2021 | Page 51

APHL 2021 Poster Abstracts and radiological events as well that may occur in the future .

Presenter : Karen Stiles , Nebraska Public Health Laboratory , kstiles @ unmc . edu

Parallel SARS-CoV-2 Detection and Genomic Characterization : A Model for Variant Surveillance in Clinical and Public Health Laboratories

R . Stinnett ¹ , A . Steyer ² , B . Briggs ¹ , H . Xie ¹ , R . Schlaberg ¹ , D . Wolk ² ;

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IDbyDNA , Inc ., Salt Lake City , UT , ² Geisinger Diagnostic Medicine Institute , Danville , PA

Background : The emergence of SARS-CoV-2 variants that may cause increased transmissibility , pathogenicity , testing errors , or vaccine breakthrough underscores the importance of nationally-distributed sequencing capacity . Virus isolation for molecular surveillance is ideal , but viral culture facilities in clinical and public health laboratories are rare . Nevertheless , clinical samples are a potential resource for molecular epidemiological analysis . We present a workflow for parallel detection and genomic analysis of SARS-CoV-2 in clinical samples from a large integrated delivery healthcare network laboratory .

Methods : Nasopharyngeal and nasal turbinate swabs in UTM were collected from patients with laboratory-documented SARS-CoV-2 infections between April and June 2020 . Residual primary samples ( n = 22 ) that tested positive with one or more EUA assays : Luminex ARIES SARS-CoV-2 ( 2 ); DiaSorin Simplexa COVID-19 Direct ( 4 ); or Cepheid Xpert Xpress SARS CoV-2 ( 16 ) were frozen ( -80 ^◦ C ) in Geisinger ’ s MyMicrobiome biorepository . Samples were selected for variety in Ct value , mixed infections , and geographic location , then submitted to an independent reference laboratory ( IDbyDNA , Salt Lake City , UT ) for RNA-sequencing analysis . Following manual RNA extraction , libraries were either sequenced directly or enriched with the Respiratory Pathogen ID / AMR Enrichment Kit ( RPIP ; Illumina , IDbyDNA ) and sequenced on a MiniSeq ( Illumina ). Bioinformatics analysis was performed via the Explify RPIP app ( Illumina BaseSpace ).

Results : Targeted enrichment sequencing detected SARS-CoV-2 in 21 / 22 ( 95 %) of samples . Predicate testing of the sample not detected by sequencing suggested low abundance ( Cepheid ; E Ct = 37 , N2 Ct = 41 ). Genome coverage of ≥ 94 % was observed for 6 / 21 samples all with an observed mean Ct of ≤ 30 . Targeted enrichment increased reads generated by > 25,000 fold in the five samples where calculation was possible and enabled detection in 16 additional samples compared to shotgun RNA-sequencing . Phylogenetic analysis and lineage determination were performed on six samples with acceptable genome coverage . Metagenomic sequencing also detected pathogens in 33 % of co-infection samples , including RSV , HuCoV229 , and HuMPV , consistent with predicate results from the BioFire Film Array RP2.1 EUA .

Conclusions : We propose a model for parallel SARS-CoV-2 detection and generation of high-quality genomic data for molecular surveillance in clinical microbiology and public health laboratories . Enhanced sequencing capacity will be critical to support active surveillance for variants of concern , especially potential viral immune escape variants associated with vaccine failures or variants associated with testing failure , should they arise .

Presenter : Rita Stinnett , IDbyDNA , rstinnett @ idbydna . com

Fully Automated Rapid Whole Genome Sequencing of SARS-CoV-2

P . Thwar Krishnan , S . Zhang , S . Shokralla , S . Lee , D . Tran , A . Allred and R . Khaksar , Clear Labs , San Carlos , CA

Background : Effective genomic surveillance during a pandemic such as COVID-19 requires rapid and high-throughput solutions to identify and subsequently track variants with higher transmissibility , virulence , vaccine-escape variants or even novel variants whose functional consequences have yet to be elucidated . WGS is able to provide more comprehensive information and is more useful in resolving such variants compared with other approaches . However , manual WGS workflows are time-consuming , labor-intensive and require specialized expertise for analysis which severely limits the practical application of WGS in public health .

Methods : Purified SARS CoV-2 RNA from 32 positive clinical nasopharyngeal swab specimens are loaded directly onto our Clear Dx platform . The Clear Dx platform is custom built with thermocyclers , sequencer and other workflow accessories housed inside a Hamilton STAR liquid handling robot and is controlled using custom instrument software . The automated WGS workflow leverages the tiled two-pool ARTIC v3 primer panel to capture and amplify targeted regions from the cDNA of the respective RNA transcripts , which were further barcoded and pooled . They were then subjected to library preparation steps customized for Oxford nanopore sequencing technology and sequenced on the sequencer . Real-time basecalling is done using a built-in data processing toolkit called Guppy , while barcode classification of sequencing reads and assembly of the consensus genome sequences were done using a modified ARTIC bioinformatic pipeline .

Results : We have developed a fully automated platform , Clear Dx , that automates all the steps of the WGS workflow with no human touch points after the start . The total run time from RNA extracts to assembled sequences for all samples takes about 20 hours . In one such run in which 32 real world clinical samples were sequenced simultaneously , we achieved a sequencing output of 4.49 million reads , of which 70 % were reliably assigned to a sample barcode . The final assembled SARS-CoV-2 genomes had a median sequencing depth of 862x and median genome coverage of 99.6 %. Since December , between our lab and our customer labs , over 8,000 SARS CoV-2 genomes have been sequenced on Clear Dx WGS platform .

Conclusions : The Clear Dx automated WGS platform , with its rapid sample-to-assembled genome sequences automation , provides an efficient tool to meet the national level public health needs such as tracking new variants and monitoring transmission patterns as well as state & local public health needs such as resolving infection clusters , outbreaks and identifying super spreader events almost in real-time . Additionally , it reduces the personnel needs for wet lab and bioinformatic analysis in public health labs and allows them to focus on the most critical epidemiological analysis .

Presenter : Prasanna Thwar Krishnan , Clear Labs , prasanna . thwar @ clearlabs . com

COVID-19