APHL 2021 Poster Abstracts
Methods : Our assay is based on reverse transcription PCR with end point analysis on matrix assisted laser desorption ionization time-of-flight mass spectrometry ( MALDI-TOF MS ). This combined platform eliminates the need for fluorescence-based detection and harnesses high sensitivities from both PCR and MALDI-TOF MS technologies . Following PCR amplification of regions on the spike gene and product cleanup , single stranded-primer extension reaction , using well-designed , target-specific primers are able to differentiate 14 mutations from consensus sequences after detection by MALDI-TOF MS .
Results : Our assay is a single-well reaction and can be run in 96- or 384-well PCR plates . Using synthetic virus RNA as quantitative control ( B . 1.1.7 _ 710528 , Twist Bioscience ), our assay demonstrated excellent sensitivity with the limit of detection ( LOD ) per reaction to 6.25 copies for K417T , 12.5 copies for P681H , K417N , L452R and I692 , 25 copies for S13I and 250 copies for other mutations . A total of 1,097 positive SARS-CoV-2 patient samples received between late December 2020 to the middle of April 2021 , were extracted and tested . Of these , 399 carried at least one mutation from the S gene . 128 possible B . 1.1.7 ( concurrent N501Y , P681H , 69 / 70 and Y144 deletion ), 166 possible B . 1.427 ( L452R ) and B . 1.429 ( concurrent L452R , S13I and W152C ) variants , three P . 1 ( Gamma *, concurrent N501Y , K417T and E484K ) and one possible B . 1351 ( Beta *, concurrent N501Y , K417N and E484K ). The first three VOCs samples , if available , and each type of other mutations were confirmed by Sanger sequencing except I692V . The B . 1.1.7 showed a rapid increase since the beginning of April , 2021 .
Conclusions : This novel assay demonstrated its unique utility with low cost and fast turnaround time to identify known SARS-CoV-2 variants during pandemics . We conclude that this novel assay can be used for rapid screening of positive SARS-CoV-2 cases to effectively monitor the prevalence of clinically-important variants in the population . Alternatively , it can also be used as a front-end screening test for whole genome next-generation-sequencing . With more widespread adoption , this assay can be used as a first-line diagnostic test to simultaneously detect SARS-CoV-2 infection and identify known variants .
* In the original poster and abstract , these were identified with country names . Per WHO naming conventions that were published in May 2021 , these have been changed .
Presenter : Yongbao Wang , National Jewish Health , wangyongbao @ njhealth . org provides the QC metrics required to pass or fail consensus genomes 2 ) visualizes mean , average , single sequence and multi-sequence coverage at each reference position 3 ) provides insight to primer sequence and primer failure regions 4 ) evaluates and recommends sequences that pass or fail QC thresholds and 5 ) sorts finalized consensus sequences into a final directory for further analyses including Pangolin variant discovery and Nextstrain phylogenetic analysis . Additional features such as quality score at each reference position may also be added . In summary , we have developed an interactive visualization dashboard for QC of SARS-CoV-2 genomic data and adequacy of the data for advanced bioinformatics analysis . The lab is currently working on obtaining the necessary approvals for making this tool public .
Presenter : Jade Wang , New York City Public Health Laboratory , jwang7 @ health . nyc . gov
COVID-19
Interactive Data Visualization Dashboard for Performing Quality Control of SARS-CoV-2 Genomic Sequences in Bulk
J . Wang , E . Gonzalez , M . Chowdhury , A . Khan , M . Hahn , S . Hughes and J . Rakeman , New York City Public Health Laboratory , New York City , NY
In early March , 2020 the first case of SARS-CoV-2 was identified in New York City ( NYC ). The New York City Public Health Laboratory ( NYC PHL ) implemented WGS of SARS-CoV-2 and to date has sequenced over 2,000 SARS-CoV-2 genomes . To ensure that sequencing data was of adequate quality for advanced bioinformatics analysis , and performing quality control ( QC ) of large datasets consisting of 50 + genomic sequences per analysis , an automated analysis tool needed to be developed . To address these challenges , we developed an interactive R Shiny application that 1 )
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Summer 2021 LAB MATTERS 51 |