Plenary session 5

Nick Loman, (University of Birmingham): Stopping outbreaks becoming epidemics. The answer is “No” (find out what this means at the end of this talk). MinIOn has been everywhere in the last year,

“I would sequence, on a boat, with a goat, on a plane, on a train, donw some holes, at the poles, in outer space, at quite a pace.”

Ebola: 2015 London Calling presentations about Ebola sequencing in Guinea (Quick et al 2016). Loads of intersting epidemiological studies came out of, or followed on from, that work. Data sharing was evidently important in making this work – proper open access. Identify cross-border transmission, identify links between cases, identify flare-ups, demonstrate delayed transmission. See Nature 2017. But even with all this effort there was a significant delay in response to this outbreak “Outbreaks are inevitable, epidemics are not”

Zika: Said he’d sequence 750 genomes on MinION – on a bus (Faria pybus loman in Genome Medicine). But almost impossibly low viral levels make diagnostics “challenging”. Two main problems 1) lots of boring DNA e.g. host, E.coli, 2) MinION requires large numbers of molecules i.e. 100ng DNA. PCR, WGA, capture all help but add time and complexity – PCR probably still the easiest method. Josh spent a few months after Zika trip working on Primal Scheme for PCR tiling. Can now get near complete Zika genomes direct from clinical samples.

Yellow fever: Large outbreak in Brazil caused 200 deaths and fear of transmission if it gets into Aedes mosquito. Josh ran Primal Scheme design, ordered primers, sent to Brazil for processing by local team. 5-6 genomes per flowcell with 1D native barcoding kit, R9.4, 1-4 million reads per flowcell of 500bp amplicons. Software pipelines deployed in Docker for easy deployment.

Long-reads: Segway to long-reads via antibiotic resistance (see picture below). Unfortunately not quite got to 1Mb read just yet. Big improvement is moving from phenol:choloform extraction to agarose plug extraction and prep. The whole E.coli genome in 7 reads.  Human genome data got to a 950kb read..we’re getting close to a finished genome on a flowcell.

Wishlist: local basecalling, no more file format changes (please), lower cost low throughput flowcells, ultra low-input prep. Can we please do some sequencing in the NHS this year or next!

Lightning talks

• Niranjan Nagaranjan: exploring gut resistomes with nanopore sequencing. Antibiotics resistance is the “climate change” of the medical community. Large projects ongoing (GIS-TTSH collaboration, CaPES) what underlies protection against colonisation, evolution of resistome, strain dynamics, etc. Using a mock community and running patient samples to understand how to unravel strain genomes. Fragmented Illumina plasmifd assemblies resolved into two closed plasmids.
• Michael Boemo (University Oxford): Detection of base  analogues with MinION. Studying replication dynamics with Thymidine analogues. A BrdU pulse followed by IdU generates base analogue regions for MinION detection to infer origins and termination sites for replication.
• Celine Bigot (CNRGH): Pathotrack project – identifying biological threats (air, water, etc) with a priori methods. Running Illumina Ion Torrent and MinION NGS. The control samples being tested includes 10 bacterial and 1 fungal species (still a work in progress). Action plan consists of testing and comparing 3 NGS technologies to assess reproducibility, sensitivity and specificity of methods. WIMP analysis shows it is possible to ID all species in control in just 5 hours.
• Scott Gigante (Walter and Eliza Hall Institute): in-house training of the nanonet local basecaller. Aiming to get methylation prediction from neural network, not HMM. Training is a black art – want to make predictions that help improve this process. Able to get 99% of CpGs called correctly.
• Ben Matern (Maastricht UMC): ID of human leukocyte antigen splice variants by MinION cDNA-Seq. Looking at HLA which presents antigens to immune cells, does funny things like alternative splicing! Using GMAP to detect exon skipping and other splicing anomalies from MinION data. Calculating expression profiles based on the exons detected, cluster and quantify, and understand variation in “normal” splicing of HLA. Aiming to test with direct RNA-Seq,
• Benjamin Istace (Genoscope): Large eukaryotic genome assembly using long-reads. Studying the banana genome. 100 million tons per year. Almost all edible species come from two ancestral genomes. Sequenced and assembled two accessions from long-reads via Pippin Prep size selected DNA. Got 22Gb of data with 18kb average read length = contig N50 of 1.85Mb.
• Matthew McCabe (teagasc): Sequencing of BRD viruses using rapid sequencing kit: BRD the leading cause of cattle mobidity and mortality in USA/Ireland, caused by DNA and RNA viruses, cause lung damage, current tests by viral qPCR. Want to make a test that can be run on the farm. Presenting data from 3 samples combined for prep and sequencing. Almost no detectable nucleic acid but made rapid library and ran on Nanopore – 7000 reads! Detected expected culprits – including nile crocodile pox virus!
• Libby Snell (ONT): Direct RNA-seq. Sequence RNA – “simples!”. Perhaps the most exciting development in RNA analysis (Here’s my post from late last year). Currently takes 2 hours, moving to rapid prep in just 30 minutes by ligating RNA protein motor on polyA tail, wash and load! Hoping that this will become the standard for RNA-Seq very soon. Showed data on ERCC, Lexogen SIRVs. Yeast sequencing looks good, some room for improvement, will be Voltrax compatible.