Dan Turner @DanTurnez: an overview of LamPORE, the new nanopore based assay for the detection of SARS-CoV-2 at #nanoporeconf. A thread:
DT: Why is the LamPORE, sequencing-based assay useful for SARS-CoV-2 detection? We think about bottlenecks: the ability to analyse very small or very large numbers of samples with a small footprint, rapid results, and efficient, precise analysis of known pathogens #nanoporeconf
DT: With RT-PCR, performing a test in duplicate or triplicate is common. If sequencing is used as an output, the precise nature of the readout means that a 96 well plate can do the full 96 samples #nanoporeconf #LamPORE
DT: the conversation is shifting from a focus on Dx of symptomatic people or their contacts, to the idea of screening larger cohorts of people. For that we need very large capacity #nanoporeconf #LamPORE
DT: The LamPORE assay is based on LAMP+rapid library prep+sequencing. LAMP: loop-mediated isothermal amplification, a fast, easy and reliable amplification method – but coupled with sequencing as a digital readout instead of colour change #nanoporeconf 
LAMP is an excellent amplification method, but colour-based readout can be ambiguous. By sequencing we can get a much more specific assay #nanoporeconf #LamPORE
DT: LAMP for LamPORE: single temperature (60-65’C) for 30 mins, 10bp barcode added to FIP primer. @nanopore sequencing barcode added during library prep. This dual barcoding enables substantial multiplexing – 1,152 samples on a single flow cell #nanoporeconf
DT: LAMP amplifies multiple targets ~150-180bp each. LamPORE targets: ORF1a, nucleocapsid (N) and envelope (E) genes. 3 targets increase detection sensitivity, even in degraded samples. These regions are common across strains, not in other resp viruses/human RNA #nanoporeconf
DT: a fourth target: human beta actin. Controls for sampling, RNA extraction, reverse transcription, and amplification - detect errors in sample collection and preparation #LamPORE #nanoporeconf
DT: The LamPORE amplicons are concatemeric reads containing multimers of the LAMP barcode and the target region. 70% of reads have both barcodes and the target region and are used in the analysis pipeline #LamPORE #nanoporeconf
DT: we picked the 8 most sensitive LAMP barcodes, used with 12 RBK barcodes for 96 combinations. (Dual barcoding with 12 rapid barcodes gets to 1,152 samples in one FC). We’re expanding further to 96 LAMP + 96 RBK => 9,216 #LamPORE #nanoporeconf
DT: a development challenge is to call true positives and avoid false positives. This ROC curve & F1 scores look at sensitivity and specificity and, allow us to set the correct read-count threshold, which for this expt is between 50-400 reads per sample #nanoporeconf #LamPORE
DT: development results: 79/80 qPCR positives confirmed, missing only one sample at Ct 38. Highest Ct detected was 37. So this is a v. sensitive assay indeed.
Negatives also confirmed, which tested +ve for human actin as sampling, amplification and prep control #nanoporeconf
Negatives also confirmed, which tested +ve for human actin as sampling, amplification and prep control #nanoporeconf
DT: LamPORE is being kitted and is now being evaluated by independent third parties, with positive early results.
Updates will be posted at nanoporetech.com/LamPORE #nanoporeconf
Updates will be posted at nanoporetech.com/LamPORE #nanoporeconf
DT: we’re looking at enriching COVID-19 host response genes using adaptive sampling. 256 regions including ACE2, TMPRSS2, MHC, and GWAS SNPs from COVID19 Host Genetics Initiative – sufficient coverage with one MinION FC #nanoporeconf
DT: we’re looking at enriching COVID-19 host response genes using adaptive sampling. 256 regions including ACE2, TMPRSS2, MHC, and GWAS SNPs from COVID19 Host Genetics Initiative – sufficient coverage with one MinION FC #nanoporeconf
DT: for direct SARS-CoV-2 detection from saliva, enzymatic treatment followed by heat inactivation step. Preliminary results are promising #nanoporeconf #LamPORE
DT: LamPORE for ‘influenza-like illness’ panel: a sneak preview of 6 virus RNAs plus total human RNA incl. actin control, still in development. These have been multiplexed #nanoporeconf
DT: Thanks to all teams at ONT especially Apps team who conceptualized, designed and tested the LamPORE lab and analysis workflows @Grounded_circus @sisseljuul @eoghh @bowerlauwer #nanoporeconf
