APHL 2021 Poster Abstracts
COVID-19
Implementation of Tracking Database and Automation to Increase SARS-CoV-2 Sequencing in Michigan
K . Wells , R . Kramer , A . Schooley , H . Blankenship and M . Soehnlen , Michigan Department of Health and Human Services , Lansing , MI
Background : SARS-CoV-2 is responsible for a global pandemic infecting millions of people worldwide over the past year . Since Michigan ’ s first detected case on March 10 , 2020 , SARS-CoV-2 has infected more than 600,000 Michiganders . WGS has played a critical role in the pandemic response through outbreak and variant tracing as well as monitoring for new mutations . The need for high throughput WGS arose due to the severity of the pandemic to ensure that a representative sample of all positive infections are sequenced to obtain an understanding of the virus in Michigan .
Methods : Michigan Department of Health and Human Services ( MDHHS ) have performed WGS on positive diagnostic samples tested at MDHHS as well as specimens sent in by clinical and commercial labs around the state . Each sample is assessed based on Ct Value through screening by CDC or Thermofisher TaqPath PCR . A subset of 472 samples were sequenced and selected to determine the Ct range that would yield > 94 % genome coverage to establish a screening threshold . All samples received are entered into a database along with the Ct values and then sorted by Ct . Those meeting the threshold move on to sequencing using one of the following combinations of Library Prep and platforms : Nextera XT / Tecan / NextSeq or Nextera Flex / Manual / MiSeq . Initial protocol development focused on manually prepping 48 samples for MiSeq . A Tecan Freedom EVO was programmed to process cDNA and library prep of 96 well plates to increase capacity and throughput .
Results : Since March 2020 , MDHHS has screened 8600 SARS- CoV-2 samples for sequencing , which resulted in over 8000 sequences and 4132 published to repositories . A comparison of Ct values and genome coverage for 472 samples determined that 72 % of samples below a 30 Ct resulted in > 94 % genome coverage . Implementation of the database allows samples to be quickly sorted into batches based upon the Ct criteria . It also allows for sample traceability by providing the capability to sort and search by run , freezer storage , and sample number . Since implementing automation , 5484 samples have been sequenced using this method . The automated system allowed for increased capacity to identify the introduction of variant strains into the Michigan population . Since identification of variants in Michigan , the number samples sequenced by Tecan and NextSeq increased from 190 per week to 570 per week to further increase capacity .
Conclusions : As a result of the staggering number of positive samples that required sequencing , it was essential to have a method to sort and organize samples . The database in conjunction with automation played key roles in sequencing . With the high throughput method , we were able to eliminate a large backlog of samples , decrease turnaround time , and begin sequencing in real time .
Presenter : Heather Blankenship , Michigan Department of Health and Human Services , blankenship @ michigan . gov
Maintaining Whole Genome Sequencing During the COVID-19 Pandemic : Improving the Cost and Efficiency of WGS with High-throughput Instrumentation
M . Dickinson , L . Mingle , K . Mitchell , D . Baker , A . Kidney , A . Sossei , K . Cummings , S . Gubbala , E . Nazarian , L . Thompson , S . Wirth , M . Shudt , W . Haas and K . Musser , New York State Department of Health / Wadsworth Center , Albany , NY
The Wadsworth Center Bacteriology Laboratory , as an Antimicrobial Resistance Lab Network Regional Lab , PulseNet Area Lab and GenomeTrakr Lab , is performing WGS on a large scale . Additionally , the WGS workload continues to increase as WGS is adopted for novel applications , such as state outbreak investigations for Legionella pneumophila and healthcare-associated infections . We compared the MagNAPure 24 , QIAcube HT , and the QIAcube Classic extractors . We evaluated the total costs of WGS , including DNA extraction , library preparation and sequencing . Improvements to workflow and a WGS testing log were developed . Efficiency was measured by hands-on time and overall run time of the instruments . Sequencing analysis was performed to compare pipeline results including genome assemblies , impact of sequencing diverse bacteria in one run , percentages of mapped reads and of bases covered by reads , and the number of single nucleotide variants ( SNV ) detected .
The most cost effective workflow combination for our lab was : batching of 80-90 samples with QIAcube HT DNA extraction , Nextera DNA Flex library prep ( using 1 / 4 of the recommended volume ), and sequencing on the Illumina NextSeq ($ 8.57 / extraction + $ 68.00 / prep & sequencing =$ 76.57 / sample ). The DNA extraction cost / sample when using the QIAcube classic is ~$ 10.00 , the MagNA Pure24 cost / sample is ~$ 20.00 . The QIAcube HT requires the least amount of bench time ( 3.75 h / 96 samples ). To compare the Nextera XT with the Nextera DNA Flex Library Preparation Kits , DNA was extracted from various bacteria , libraries were prepared with either kit , and WGS was performed . In most cases , data generated from Nextera DNA Flex libraries resulted in higher quality genome assemblies . When reads were mapped to appropriate reference genomes , both library prep methods resulted in similar percentages of mapped reads and bases covered by reads . Finally , the number of SNV was comparable for both methods . This study demonstrated that the combined use of the QIAcube HT , Nextera DNA Flex library prep kit and the Illumina NextSeq platform streamlined sequencing of the WGS requests across multiple laboratory units . This study demonstrated that the implementation of this workflow maximized cost-effectiveness and decreased turnaround time . In 2020 , the COVID-19 pandemic strained both our staffing and instrumentation resources as staff members were reassigned to the COVID-19 response . The previous validation and implementation of batching of all bacteria with this workflow allowed us to continue our sequencing responsibilities even with decreased staffing . Highvolume public health laboratories should consider implementing these methods to aid in meeting testing requirements within budgetary restrictions in a timely manner while reducing staff burden .
Presenter : Michelle Dickinson , New York State Department of Health / Wadsworth Center , michelle . dickinson @ health . ny . gov
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LAB MATTERS Summer 2021 |
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