Lab Matters Fall 2023 | Page 80

APHL 2023 POSTER ABSTRACTS

determine how the removal of PqsE in this system alters QS gene expression and if it renders these strains non-pathogenic . We hypothesize that the PqsE-RhlR interaction is crucial for QS and disease progression . Our results have identified CF isolates with elevated levels of RhlR and PqsE expression , which correlates to an increase in QS gene expression . By combining transcriptional and genomic data in P . aeruginosa isolates from CF patients we can better understand the development of acute and chronic infections and potentially identify targets for future therapies and improve the health outcomes for those with CF .

Presenter : Elizabeth Knorr , elizabeth . knorr2 @ gmail . com

Whole Genome Sequencing of Influenza A and B : Assessment of Different cDNA Preparation Strategies

V . Nguyen , K . Castro-Vital , M . Hetherington-Rauth , S . Baird , A . Rossheim and S . Matzinger ; Colorado Department of Public Health and Environment

Background : High throughput whole genome sequencing ( WGS ) for influenza A / B during peak influenza season allows for rapid response to emerging variants to assist in the public health response for ongoing surveillance . The current national influenza A / B WGS surveillance system effort employs an amplicon-based approach . Here , we investigated the usefulness of specific adaptations to the nationally approved protocol for increased flexibility for surge cases , such as increased sample volume and supply chain issues , as well as more cost-effective options for cDNA preparations . We tested three areas where cDNA preparation could be optimized . ( 1 ) We compared the use of two reverse transcriptases — LunaScript ( LS ) and SuperScript III . LS has been shown to have higher fidelity than SSIII , but it is more expensive . ( 2 ) We tested pooling A and B primer sets together , rather than using each primer set separately , to see if we could avoid the need to determine each sample ’ s subtype by PCR before WGS . ( 3 ) We assessed whether using a lower primer concentration resulted in similar WGS results to higher primer concentrations , which would reduce reagent costs . We assessed the effect of these three optimization tests by evaluating the average sequencing depth and percent genome coverage . Methods and Results : We sequenced 10 clinical A samples with controls in duplicates using LS vs SSIII . The results showed > 97 % coverage on all eight gene segments when using SSIII compared to inconsistent coverage with LS . SSIII also showed higher sequencing depth . To determine whether pooled primer sets are effective in providing the same percent coverage and depth as unpooled primer sets , three different cDNA preparations were tested : ( 1 ) Only A primers , ( 2 ) Only B primers , and ( 3 ) Pooling A / B primers together . Unpooled primer sets showed higher sequencing depth compared to pooled primer sets . However , the reduced sequencing depth from the pooled primer sets did not affect overall sequence quality and was well within national sequence quality metrics guidelines . To assess the effect of primer set dilution , A / B primers were diluted twice over dilutions recommended in the current protocol utilized for national surveillance efforts . Using a mixture of influenza A / B clinical samples , the sequencing results of the lower limit of dilution still produced > 97 % coverage on all eight segments . Influenza A samples sequenced better , in terms of coverage and depth , than Influenza B samples . Conclusion : Our results demonstrate that to save time and resources , subtyping can be skipped and samples can be quickly sequenced using pooled primers . To be more cost effective , without diminishing quality , SSIII performs better compared to another commonly utilized enzyme , LunaScript . Finally , appropriately diluted primers may provide a cost-saving alternative that should be explored further .

Presenter : Van Nguyen , van . nguyen @ state . co . us

Whole Genome Sequencing of Mixed Pathogen Runs to Reduce Turnaround Time and Costs per Sample

D . Mallal , V . Nguyen , A . Alford , A . Lo , M . Heatherington-Rauth , S . Matzinger , A . Rossheim ; Colorado Department of Public Health and Environment

The demand for whole genome sequencing ( WGS ) as an advanced molecular detection method for outbreak detection and pathogen characterization has increased , especially in the post-COVID era . With this increase in demand , there is a need to be able to quickly and robustly onboard new pathogens while also being cognizant of cost and turnaround time . The Colorado Department of Public Health and Environment ( CDPHE ) Laboratory rapidly expanded sequencing capacity , adding multiple pathogens of public health concern to sequencing repertoire . In order to do this effectively without impacting turnaround time of routine sequencing efforts , including COVID surveillance and PulseNet , we implemented sequencing multiple pathogens on a single Illumina MiSeq run , a method supported by the PulseNet Network for Norovirus WGS . By expanding mixed pathogen sequencing runs to include additional viral organisms , we significantly decreased the cost of sequencing and turnaround time per sample . The maximum loading capacity of a typical PulseNet run on the Illumina MiSeq platform that includes Shigella , E . coli , or Vibrio is 100MB (~ 20 Salmonella , E . coli , or Shigella isolates ) for a v2 500 cycle kit , or 175MB (~ 35 Salmonella , E . coli , or Shigella isolates ) for a v3 600 cycle kit . Although PulseNet confirmed mixed pathogen runs are successful when adding in amplicon sequencing of Norovirus , we found that other viral organisms , such as influenza , can also be appended to a sequencing run without affecting the quality of sequence coverage . Our strategy for sequencing viral genomes , including influenza , mpox , norovirus and RSV ( Respiratory syncytial virus ) on mixed pathogen sequencing runs is to first generate amplicon-based cDNA followed by the Illumina DNA library preparation method . Next , these amplicons are spiked into a full PulseNet run , varying the volume spiked in based on genome size and depth of coverage requirements of the virus . We successfully performed WGS on 48 influenza ( genome size 13,500 bps ) samples in addition to a full PulseNet run on both v2 and v3 chemistries without sacrificing quality or depth of coverage of either sample type ( For example , influenza maintains an average of > 100x coverage depth , and bacterial isolates maintain a > 40x average De Novo coverage ). This procedure reduces the loading cost per sample from ~$ 67 ( 20 bacterial samples on a v2 kit ) to ~$ 20 per sample ( 20 bacterial samples plus 48 influenza samples on a v2 kit ). For mpox ( genome size 190,000 bps ), which has a much larger genome than influenza , we included 16 samples on routine PulseNet runs , and observed excellent coverage ( average > 400x coverage ) of the viral amplicons without any decrease in quality metrics for the bacterial samples on the run . We successfully demonstrated how mixed pathogen sequencing runs can reduce the cost per sample and overall turnaround time while maintaining high quality .

Presenter : Daniel Mallal , daniel . mallal @ state . co . us