On 30 November–1 December 2017, scientists from the Nanopore Community will meet in New York to discuss their work. The agenda will include plenary and breakout sessions, lightning talks, poster sessions, conference dinner.
The pre-meeting workshop will take place on 29 November.
- Innovators' incentive: SOLD OUT
- Limited offer: $650 (Offer expired 16th October)
- Poster rate: SOLD OUT
- Standard rate: SOLD OUT
- Pre-meeting workshop: SOLD OUT*
* Available to members of the Nanopore Community only. This one-day workshop is an additional cost.
University of Birmingham
The Sequencing Singularity?
Sequencing is poised to disrupt clinical practice. Whole swathes of diagnostic tests may eventually be replaced with a single assay - sequencing - as we reach the "sequencing singularity". I will review recent advances in the use of nanopore sequencing for clinical microbiology and human genetics, including our collaborations on viral and bacterial diagnostic sequencing, real-time surveillance, direct RNA and human whole-genome sequencing, and discuss the opportunities and barriers around moving to sequencing as a routine test in the clinic.
Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples, Jan 2017, BioRxiv
Human Reference on Oxford Nanopore MinION, Dec 2016, GitHub, https://github.com/nanopore-wgs-consortium/NA12878
De novo Identification of DNA Modifications Enabled by Genome-Guided Nanopore Signal Processing, Dec 2016, BioRxiv, doi: https://doi.org/10.1101/094672
Real-time, portable genome sequencing for Ebola surveillance, Feb 2016, Nature, https://doi.org/10.1038/nature16996.
A complete bacterial genome assembled de novo using only nanopore sequencing data, Apr 2016, Nature Methods, https://doi.org/10.1038/nmeth.3444
Epidemic establishment and cryptic transmission of Zika virus in Brazil and the Americas, Feb 2017, BioRxiv, doi: https://doi.org/10.1101/105171Agenda
Bjarni V. Halldórsson
Using long read Oxford Nanopore sequencing for population scale human genetics
Short read sequencing has allowed exact identification and calling of single nucleotide polymorphisms and short indels and has substantially advanced the study of human populations. However, with this technology it is not possible to identify complex structural variants and polymorphisms in repeated or low-complexity regions. Here, we describe the results obtained from our current long read sequencing project of 100 Icelanders with Oxford Nanopore technology. These individuals had already been deep sequenced with Illumina technology, both for variant calling and estimation of DNA methylation based on treatment with bisulphite. We describe the concordance between the different sequencing platforms both for variant and methylation calling and describe some of the additional information provided by the long read data.
Bjarni received his Bachelor of Science (BSc) degree in mathematics from the University of Iceland in 1996, and completed a PhD degree in Algorithms, Combinatorics and Optimization from Carnegie Mellon University in 2001. He was a research scientist at Celera Genomics from 2001 until he joined deCODE genetics in 2004. He has been an associate professor of Biomedical Engineering at Reykjavík University from 2006. Since 2013 he has been Head of Sequence Analysis at deCODE genetics.Agenda
UPMC – Pierre et Marie Curie University
Stéphane Le Crom
RNA sequencing and data analysis using the MinION system
Our core facility focuses on eukaryote functional genomics. Since 2010 we worked on Illumina high throughput sequencers providing RNA-seq and ChIP-seq results to our users. As estimation of transcript isoform abundance is a real challenge, we moved last year to Oxford Nanopore sequencing technology in order to get full length transcripts sequences.
During this talk we will present the results we obtained on MinION using our mouse model of peripheral nervous system development. We performed barcoded runs using 2D and 1D chemistry on R9 and R9.4 MinION flowcells. We examined barcode influence on sequencing yield and we tested the new cDNA kit.
We will also present the computer infrastructure and workflow we set-up for data analysis. We developed the ToulligQC software for MinION run quality control dedicated to RNA-Seq experiments. We also performed tests for transcripts data alignments and obtained differential analysis targets using our mouse model.
Stéphane Le Crom is a Professor of Genomics at Pierre et Marie Curie University (UPMC, Paris, France). His research interests focus on functional genomics, quality controlled and reproducible bioinformatics analysis and core facilities management. Since 2014 he is responsible for a network of life sciences and health facilities at UPMC.
He is also the scientific head of the Genomics facility at the École normale supérieure Biology Institute (IBENS) in Paris. This facility has been created in 1999 for providing access to functional genomics technologies with the main objectives to help laboratories managing their genomic projects, disseminate genome-wide approaches among the scientific community, and develop competence and resources in bioinformatics analyses of functional genomic data.
Can Nanopore sequencing finally finish the human genome?
A complete and accurate genome sequence forms the basis of all downstream genomic analyses. However, even the human reference genome remains incomplete, which affects the quality of experiments and can mask true genomic variations. For most other species, high-quality reference genomes do not exist. Long-read sequencing technologies, like Oxford Nanopore, have begun to correct this deficiency and are enabling the automated reconstruction of reference-quality genomes at relatively low cost. In a collaborative effort, we sequenced the NA12878 human genome and assembled using Canu. The sequencing set includes 5-fold coverage of ‘ultra-long’ reads with an N50 of >100 kbp and max length >800 kbp. Despite the low coverage, the assembly contiguity (NG50 ~6.4 Mb) exceeds that of similar coverage assemblies using other long-read technologies. The ultra-long reads enable the complete phasing across the MHC and close large gaps (>50 kbp) in the existing reference assembly. Additionally, we model expected assembly contiguity and predict 30-fold coverage of ultra-long sequences can exceed a 40 Mbp NG50 and match the contiguity of the current reference. Further combination of these technologies with complementary scaffolding and phasing approaches such as chromatin conformation capture (Hi-C) may soon enable the complete reconstruction of vertebrate haplotypes.
Canu source code and pre-compiled binaries are freely available under a GPLv2 license from https://github.com/marbl/canu. The complete NA12878 dataset including assembly and raw signal is available as an Amazon Web Services Open Dataset at: https://github.com/nanopore-wgs-consortium/NA12878. The ‘Cliveome’ is available from https://github.com/nanoporetech/ONT-HG1.
Sergey received his PhD in computer science in 2012 under the supervision of Mihai Pop at the University of Maryland. He joined the National Bioforensics Analysis Center in 2011 and was appointed as an associate principal investigator in 2014. During this time, he pioneered the use of single-molecule sequencing for the reconstruction of complete genomes. In 2015, he joined the National Human Genome Research Institute as a founding member of the Genome Informatics Section. His research focuses on the efficient analysis of large-scale genomic datasets and new methods for metagenomic analysis and assembly of high-noise single-molecule sequencing data.
Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation, March 2017, Genome Research, https://doi.org/10.1101/gr.215087.116Agenda
University of Melbourne
Assembly is still hard: challenges in genome assembly in the era of long reads
Genome assemblers are tools which aim to reconstruct an original genome from sequencing reads. A ‘perfect’ assembler would take only reads as input and output a complete, error-free genome. This goal is usually impossible with short read sequencing, but long reads from Oxford Nanopore sequencers bring it tantalisingly close.
While long reads are enormously helpful for assembly, issues remain. In this talk, I will describe the various ways an assembly can fail: sequence inaccuracy, incomplete or misassembled sequences, and lost variation. For each, I will discuss the underlying causes and what future developments are necessary to overcome them and bring us closer to perfect assemblies.
Ryan Wick received a BSc from the University of Wisconsin–Madison and a Master’s in Bioinformatics from the University of Melbourne. He currently works in the Holt Lab at the University of Melbourne’s Centre for Systems Genomics. Ryan is particularly interested in de novo assembly and is the creator of Bandage, an interactive assembly graph viewer, and Unicycler, a hybrid assembly tool for bacterial genomes.
Unicycler: resolving bacterial genome assemblies from short and long sequencing reads, Dec 2016, BioRxiv, doi: https://doi.org/10.1101/096412Agenda
Oxford Nanopore Technologies Ltd
Applications in genetics and genomics using nanopore DNA, cDNA and direct RNA sequencing
Sissel Juul joined Oxford Nanopore in the summer of 2014 to lead the company’s US Applications group, setting up a lab in New York City. Recently, the US Apps group has expanded, with the opening of a second lab, in San Francisco. The US teams utilize the unique strengths of ONT's technologies to showcase high-impact biological applications both independently, as well as with external collaborators and ONT customers. This leads to scientific papers, and posters and presentations at conferences. Prior to joining ONT, Sissel did her postdoctoral research at Duke University, NC, and has a PhD in molecular biology and nanoscience from Aarhus University, Denmark.Agenda
University of Berkeley
Real-time DNA barcoding in a remote rainforest using nanopore sequencing
Advancements in portable scientific instruments provide promising avenues to expedite field work in order to understand the diverse array of organisms that inhabit our planet. We tested the feasibility for in situ molecular analyses of endemic fauna using a portable laboratory fitting within a single backpack, in one of the world’s most imperiled biodiversity hotspots: the Ecuadorian Chocó rainforest. Here we demonstrate that nanopore sequencing can be implemented in a remote tropical field site to quickly and accurately identify species using DNA barcoding, as we generated consensus sequences for species resolution with an accuracy of >99% in less than 24 hours after collecting specimens. Overall, we establish how mobile laboratories and nanopore sequencing can help to accelerate species identification to aid in conservation efforts and be applied to research facilities in developing countries to promote local capacity building.
Aaron Pomerantz is a PhD student at UC Berkeley in the Department of Integrative Biology and National Geographic explorer. Prior to starting his doctoral program, Aaron spent two years leading expeditions as a field biologist and science reporter in the Amazon rainforest, where he became interested in applying novel technology to conduct fieldwork in remote locations, such as origami-based portable microscopes and handheld gene sequencers. Aaron continues to conduct research at field locations such as the Tambopata Research Center in Peru and in addition to exploring portable technology for fieldwork, his dissertation aims to elucidate the development and genetic mechanism of structural coloration and wing transparency in butterflies.Agenda
University of Oxford
Elucidating the expression and splicing patterns of neuropsychiatric disease genes in human brain
The Schizophrenia and Bipolar disorder risk gene CACNA1C is one of the most promising targets for future psychiatric therapeutics, with both a known function and established 'druggability'. However, its extreme length (>13kb), number of exons (>50) and high level of alternative splicing means CACNA1C isoform structure and expression is poorly understood, making it difficult to identify which gene isoforms confer disease risk and where they are expressed. We have investigated CACNA1C expression in six regions of post-mortem human brain by Nanopore sequencing of the complete coding sequence from expressed transcripts. The ability of Nanopore sequencing to define the complete exonic structure of CACNA1C allows us to identify known and novel gene isoforms, including novel exons and multi-exonic skipping events and their expression across multiple brain regions. These results are the first step in evaluating CACNA1C as a potential therapeutic target and demonstrate the power of long-read Nanopore sequencing to elucidate the true nature of expressed genes.
Mike Clark is a CJ Martin Biomedical Research Fellow based at the Department of Psychiatry, University of Oxford. His research sits at the intersection of genomics and neuroscience, utilizing a number of genomic approaches, including Nanopore sequencing, to investigate gene expression and function in the human brain and in neuropsychiatric disorders. Prior to working at the University of Oxford, Mike completed a PhD at the University of Queensland (UQ) in Australia and postdoctoral work in Australia at UQ and the Garvan Institute for Medical Research.Agenda
University of Arkansas for Medical Sciences
Transcriptional Landscapes analysis through direct RNA sequencing
Obtaining a complete genome and transcriptional landscapes of eukaryote can be a difficult task, due to a combination of highly repetitive sequences along the chromosomes and short read lengths obtained from second-generation sequencing. With long reads were generated using Oxford Nanopore (ONT), we obtained complete genome through de novo assembly. Furthermore, we generated long reads using direct RNA sequencing with ONT to investigate transcriptional landscapes and quantification. Full-length transcripts were identified through a novel approach of direct RNA-seq. This method provides accurate identification of transcriptional landscapes, including untranslated regions as well as differential gene expression quantification. Direct RNA-seq identified many polyadenylated non-coding RNAs, including rRNAs, telomere-RNA and a long noncoding RNA.
Intawat Nookaew, PhD is an Associate Professor in Department of Biomedical Informatics and Department of Physiology & Biophysics. He has experience in both experimental work and extensive computational analysis in broad domains of life. His research is focused on applied bioinformatics and systems biology. He has developed many robust frameworks and computational analysis packages to handle, analyze and integrate large-scale datasets such as multilevel omics data. He also has extensive experiences in genomics and transcriptomics aerea. He has applied the developed frameworks on several projects in diverse areas of clinical/biomedical and biotechnological research.Agenda
French National Institute for Agriculture (INRA)
Best practice to maximize throughput with Nanopore technology & de novo sequencing of bacterial strains of Xanthomonas campestris and genetic lines of Arabidopsis thaliana.
At the sequencing core facility of Toulouse (Get-PlaGe - France), we started to work with 3rd generation sequencers in 2015 with the installation of a PacBio RSII machine. Since almost three years, we have gained experience in library preparation for this type of sequencing, which is largely different from the library preparation for 2nd sequencing generation technologies (Illumina). In particular, because the native molecules are directly sequenced, a very high quality DNA is required. In addition, we assessed several optional steps in the library preparation process such as DNA reparations, fragmentation and size selection. In line with this, preliminary results for de novo sequencing obtained with the Nanopore technology and assembly of several strains of the phytopathogenic bacterial species Xanthomonas campestris isolated from natural populations of Arabidopsis thaliana will be presented. Finally, the results on the assembly of a new reference genome of A. thaliana will also be introduced.
Baptiste obtained a Master degree in Biotechnology from the University of Limoges (France) in 2010. Then, he spent 2 years at the seed company Limagrain Europe (France) to study Copy Number Variations in maize and to develop molecular markers in various crops. In 2012, he joined the Laboratory of Plant-Microbe Interactions (French National Institute for Agricultural Research) in Toulouse (France) to work on the genetics and genomics of sunflower. He especially produced the PacBio data for the sunflower reference genome that has been released in 2017. Since September 2015, he has a position in the group of Fabrice Roux to work on the ecological genomics of adaptation in plant communities. He now uses the Nanopore technology to sequence bacterial strains and different genetic lines of the model plant Arabidopsis thaliana to answer different ecological questions related to the adaptation of A. thaliana to its microbiota and plant communities.Agenda
Johns Hopkins University
Assembly of large genomes using Oxford Nanopore and Illumina data
We have recently developed hybrid assembly methods that use a combination of short and long reads to produce remarkably high-quality assemblies from whole-genome shotgun data. The new methods are integrated in the MaSuRCA assembler system. In this talk I will describe our recent algorithm developments and their use in assembling a pathogenic fungus genome (37 Mb) and the English walnut tree genome (600 Mb), both of which produced highly contiguous genome assemblies. I will also describe our plans to to use Oxford Nanopore sequencing as part of our strategy to assemble the very challenging mega-genomes of two of the largest trees on Earth: the giant sequoia, Sequoiadendron giganteum (11 Gb) and the Coast Redwood, Sequoia sempervirens (33 Gb).
Steven Salzberg is the Bloomberg Distinguished Professor of Biomedical Engineering, Computer Science, and Biostatistics and the Director of the Center for Computational Biology in the McKusick-Nathans Institute of Genetic Medicine at Johns Hopkins University. Dr. Salzberg received his bachelor's and master's degrees from Yale University, and his Ph.D. in Computer Science from Harvard University. Salzberg's lab currently focuses on next-generation sequence alignment, large-scale genome assembly, and microbiome analysis. In recent years Salzberg and his students introduced several pioneering, highly efficient systems for alignment of next-generation sequencing reads, including the Bowtie, Tophat, and Cufflinks systems, which are used by thousands of labs around the world. All of the group's software is free and open source, and their systems have been downloaded hundreds of thousands of times. In addition to his software systems, Salzberg has contributed to many genome sequencing projects, including the human genome, multiple plant and animal genomes, and many bacteria.
Dr. Salzberg has authored or co-authored over 250 publications that have garnered over 135,000 citations, and his h-index is 124. He was the 2013 winner of the Benjamin Franklin Award for Open Access in the Life Sciences. In 2001, 2014, 2015, 2016, and 2017 he was selected by Thomson Reuters/Clarivate Analytics as a Highly Cited Researcher, a compilation of the 1% most-cited researchers in the world. He also writes a widely-read science column at Forbes, http://www.forbes.com/sites/stevensalzberg.Agenda
University of East Anglia
Rapid metagenomic diagnosis of hospital acquired pneumonia
Hospital acquired pneumonia (HAP), respiratory infection developing > 48 h after hospital admission, affects 1.5% (200k) of hospital inpatients per year in the UK and has a 25-50% attributable mortality rate associated with infection. Current culture based diagnosis has sub-optimal specificity and sensitivity and is too slow (>48 hrs) to impact on patient management. Rapid diagnostics are urgently needed to improve the clinical management of HAP, to improve patient outcomes and antimicrobial stewardship. Shotgun metagenomic sequencing has the potential to change the way we diagnose HAP, with improved speed and accuracy compared to current methods.
We have developed and optimised a metagenomics pipeline for the diagnosis HAP including human DNA depletion, pathogen DNA extraction, library preparation, MinION sequencing and Epi2Me analysis.
We have applied this pipeline to >50 respiratory samples and compared the results to microbiological culture. The turnaround time from sample to pathogen and acquired resistance gene identification is approx. 6 hours, at least 42 hours faster than culture. The overall specificity and sensitivity of the method for pathogen identification compared to culture was >90%. Analysis of resistance data is still underway, however, the mecA gene was detected in all samples positive for MRSA. Real-time metagenomics has the potential to replace culture for the diagnosis of pneumonia and provides the rapid turnaround necessary for precision management of HAP patients.
Dr O'Grady gained his BSc, MRes and PhD in microbiology at the National University of Ireland Galway. He remained in Galway for his first post-doc, continuing his research in food microbiology. This was followed by a two year stint in at Beckman Coulter, developing rapid diagnostic tests for infectious diseases. Dr O’Grady then returned to academia, taking up a post-doc position at University College London on TB diagnostics. In January 2013 he was appointed Assistant Professor in Medical Microbiology at the University of East Anglia, UK and was promoted to Associate Prof in August 2016. His research continues to focus on the rapid molecular detection of pathogens, with a particular focus on rapid metagenomics sequencing based infection diagnosis.Agenda
Institute for Infectious Diseases (IFIK), University of Bern
Applications of nanopore sequencing technologies to whole genome sequencing of human viruses in the clinical setting
PCR-based detection is still the most commonly used method for routine identification of viruses of clinical importance, and this approach is often complemented by Sanger sequencing for further genotype determination. Yet, point mutations and recombination events that occur frequently in the genomes of human viruses can potentially lead to false negative results when PCR-based techniques are uniquely used. Here, we developed a set of assays based on Oxford nanopore technologies to obtain whole genomes of enteroviruses (RNA viruses) from clinical samples. We will present our conclusions about direct RNA sequencing vs. indirect sequencing of RNA (via cDNA synthesis and sequencing), in terms of costs, turnaround time, accuracy, and types of foreseen applications in the clinical setting.
Alban Ramette received a PhD degree in Natural sciences from the Swiss Federal Institute of Technology (ETHZ) Zurich Switzerland in 2002. His work mostly focused on environmental microbiology and biostatistical analyses of complex datasets (Michigan State University 2002-2005; Max-Planck Institute Bremen, Germany, 2005-2014). From 2014-2016, he worked on the epidemiology of child asthma and respiratory diseases at the Institute for Social and Preventive Medicine, Bern, Switzerland. In September 2016, he started the Bioinformatics/Biostatistics group at the Institute for Infectious Diseases, University of Bern, Switzerland. Since then, his group has focused on high-throughput sequencing solutions for clinical applications with strong emphasis on products from Oxford Nanopore Technologies. He brings expertise in NGS analysis and statistical analysis of large datasets from clinical and environmental microbiology.
Weill Cornell University
Detecting m6A in bacterial DNA
Using a tool we developed to detect base modifications from nanopore sequencing data (mCaller), we profiled methylated motifs in reference bacteria. We use nanopore sequencing to confirm known methyltransferase target motifs and validate new motifs detected by PacBio. Most non-motif sites detected by PacBio are missed by nanopore, suggesting these sites are false positives, whereas canonical target motifs missed by PacBio are identified as methylated using nanopore. We investigate the current limitations for both available single molecule methods of m6A detection and are develop a set of well-characterized reference materials for further methods development and testing.
Alexa McIntyre is a PhD student at Weill Cornell in New York City. Her research focuses on base modifications in hosts and microbes.Agenda
Johns Hopkins University
Applying Nanopore Sequencing and Real-Time Antimicrobial Resistance Gene Analysis to Address the Threat of Carbapenem-Resistant Gram-Negative Organisms
More than 700,000 people around the world die from antibiotic-resistant infections annually, translating to 1,900 deaths per day. One of the most significant antibiotic resistance threats is carbapenem-resistant Gram-negative organisms (CRO). This presentation will review methods for which Nanopore sequencing and real-time antimicrobial resistance gene analysis can help to address the threat of CRO as a diagnostic tool in the Medical Microbiology Laboratory. Applications reviewed will include detection of gastrointestinal colonization with CRO using metagenomics next-generation sequencing and real-time resistance gene analysis of whole genome sequencing data from carbapenem-resistant organisms to predict therapy. These applications of nanopore sequencing in the hospital setting has the potential to impact infection control and antimicrobial stewardship initiatives.
Patricia (Trish) Simner, PhD, D(ABMM), is an Assistant Professor of Pathology at the Johns Hopkins University School of Medicine and the Director of the Medical Bacteriology and Parasitology Laboratories at the Johns Hopkins Medical Institutions in Baltimore, MD. She completed her PhD at the University of Manitoba in Manitoba, Canada and a two year Clinical Microbiology Fellowship at the Mayo Clinic in Rochester, MN. Dr. Simner is a certified Diplomate of the American Board of Medical Microbiology. Her research has focused on understanding the epidemiology and molecular mechanisms of resistance of Gram-negative bacteria, in particular those harboring β-lactamase enzymes. With the increasing prevalence of carbapenemase-producing organism (CPO), her focus has evolved to studying novel diagnostic methods for the detection of these clinically important pathogens and understanding their mechanisms of resistance and spread in the hospital setting. For a young researcher, she is well published with greater than 60 peer-reviewed manuscripts and 9 book chapters. She was recently named as the recipient of the American Society for Microbiology (ASM) 2018 Diagnostics Young Investigator Award. Dr. Simner is currently the principal investigator for a National Institute of Health (NIH) grant which seeks to understand the molecular mechanisms of antibiotic resistance and spread amongst all CPO in a region of the US endemic for CPO. In addition, she is involved in many interdisciplinary collaborative research projects, including a Centers for Disease Control and Prevention (CDC) Epicenter grant and a NIH R01 subaward with the University of Michigan. She is also interested in novel diagnostic tools for infectious diseases and is actively involved in validating metagenomic next-generation sequencing as a diagnostic tool. Dr. Simner is a member and Advisor for the Subcommittee on Antimicrobial Susceptibility Testing for the Clinical and Laboratory Standards Institute, a member of the American Board of Medical Microbiology exam validation committee for the American College of Microbiology and a member of the Antimicrobial Resistance Surveillance Taskforce for the CDC. She is also an Editorial Board Member for the Journal of Clinical Microbiology.Agenda
University of Strasbourg
Yeast genome assembly and structural variant mapping using Nanopore sequencing
Comprehensive genomic variant maps are essential to explore genome evolution as well as its phenotypic consequences in natural populations. To date, short-read sequencing allowed to have genome- and species-wide views of mainly single nucleotide and copy number variants as we recently obtained in the Saccharomyces cerevisiae species by whole genome sequencing of 1,011 natural (http://1002genomes.u-strasbg.fr/) isolates using an Illumina technology. However, the detection of structural variants (e.g. long indels, inversions, translocations) (SVs) still poses challenges, more precisely when variants are in high complexity regions while they correspond to genetic variants underlying phenotypic variation. Emerging long-read sequencing technologies, such as Oxford Nanopore MinION sequencing, provide an unprecedented opportunity to efficiently detect these structural variants. To evaluate the performance of this technology for whole-genome assembly and SVs detection, we resequenced various genomes of natural isolates of two distinct yeast species, namely Saccharomyces cerevisiae and Dekkera bruxellensis, showing different degree of genomic complexities. Using the ONT MinION at 20x mean coverage, highly complete and contiguous assemblies have been obtained. Data generated allowed hence to accurately detect SVs, such as translocations and large inversions throughout the genomes. Among the long inserted and deleted regions, we identified those related to transposable elements and could provide a complete cartography of these elements among the sequenced isolates. Our preliminary results clearly show the value of the MinION system for screening whole genomes for complex SVs and deeply characterizing genome architecture in yeast natural populations.
Joseph Schacherer is professor of genetics and genomics at the University of Strasbourg, France. He leads an experienced research team combining people with expertise in population genomics, genetics, bioinformatics and data analysis crucial to set up high-throughput sequencing and phenotyping experiments. The group long-term goal is to use population and functional genomics to have a better insight into the rules that govern the genotype-phenotype relationship within species. He has been nominated member of the Institut Universitaire de France (IUF) in 2016 as well as a member of the University of Strasbourg Institute for Advanced Study (USIAS) in 2017.
Fournier T, Gounot JS, Freel K, Cruaud C, Lemainque A, Aury JM, Wincker P, Schacherer J, Friedrich A. High-quality de novo genome assembly of the Dekkera bruxellensis yeast using Nanopore MinION sequencing. G3 (Bethesda). 2017. 7(10):3243-3250. Istace B, Friedrich A, d'Agata L, Faye S, Payen E, Beluche O, Caradec C, Davidas S, Cruaud C, Liti G, Lemainque A, Engelen S, Wincker P, Schacherer J, Aury JM. de novo assembly and population genomic survey of natural yeast isolates with the Oxford Nanopore MinION sequencer. Gigascience. 2017. 6(2):1-13.Agenda
CReATe Fertility Center
Nanopore sequencing (MinION) for comprehensive Preimplantation Genetic Diagnosis (PGD) of chromosomal rearrangements
Carriers of balanced chromosomal rearrangements are at higher risk for reproductive failure, recurrent miscarriages or offspring with unbalanced chromosomal rearrangements. Using Preimplantation Gene Screening (PGS) to select apparently balanced euploid embryos for transfer is the current diagnostic approach for these patients. By doing PGS, patients will have higher implantation rate and lower miscarriage rate however, the carrier status of the embryos for the chromosomal translocation and possible cryptic microdeletions/duplication at breakpoint sites is difficult to detect with current NGS methods. Their main limitation is that they produce short reads of DNA sequence that may not map uniquely to repetitive regions of the human reference genome, making the detection of breakpoints difficult even with extensive high depth sequencing. Nanopore sequencing with the MinION provides a way to produce long single-molecule reads ultimately overcoming the above mentioned limitation by giving precise information of the breakpoint location and the type of rearrangement itself. The scalability, portability and also the ability for real-time data analysis makes MinION an attractive method for PGD.
We used ONT MinION sequencing to establish a protocol for preimplantation genetic diagnosis of chromosomal rearrangements in 3 couples where one of the partners is a carrier of a balanced translocation [1. t(8,22)(q24.3;q13.1), 2. t(1,2)(p34.1;p13), 3. t(6,14)(p25;q24.1) and 4. t(8;22)(q24.1;q11.2)].
We developed a bioinformatics pipeline to map the chromosomal rearrangements from the long-read data and successful breakpoint localization in all 4 translocation carriers. The mean whole genome coverage was 3x using the 1D ligation kit and a single R9.4 flowcell. Custom breakpoint PCR amplification systems were developed for each specific translocation allowing high sensitivity tests in embryos (n=21) produced by the couples.
We demonstrate for the first time application of long-read ONT MinION sequencing as an emerging solution for high resolution comprehensive clinical preimplantation genetic diagnosis of chromosomal rearrangements.Agenda
Validation of low cost, long read sequencing for crop pangenomics
The Minion is a portable, long read sequencer that can run off of a standard laptop computer. This versatility opens the possibility of genomics-guided breeding in remote and under-resourced locations. Here we validate the Minion’s application to crop genomics by creating a de novo genome assembly of the heterozygous ‘Pound 7’ clone of cacao (Theobroma cacao), and compare it to the previous two cacao reference genomes. We found that generating a high-quality assembly of a heterozygous plant genome was possible for less than $5000, in contrast to previous cacao reference genomes, which required many years and millions of dollars to produce.
Dr. Stack is a senior scientist in the applied breeding and genetics program at Mars, Incorporated. He received a B.S. degree in Computer Science from North Carolina State University and a Ph.D. in Biology from The Pennsylvania State Univeristy. Over the past 5 years he has been involved in multiple genome assembly projects related to Theobroma cacao (Chocolate) employing a wide range of sequencing technologies. He has been using MinION sequencing for the last year to de novo assembly heterozygous cultivars, and to scaffold other, highly-fragmented assemblies.Agenda
University of Aberystwyth
MinION microbiome profiling: going from on-the-go to go-to?
Our current perspective of our microbial world is heavily influenced by high throughput DNA sequencing and its limitations. Our interest in MinION sequencing has focused on developing approaches for sequencing in extreme environments to bypass some of the bottlenecks associated with sequencing in microbial ecology. In this talk I will introduce our work in extreme environments as varied as the terrestrial subsurface and Arctic glaciers. Our DeepSeq 1 and 2 experiments were conducted in the Earth’s subsurface and demonstrate the rapid generation of microbiome profiles using lightweight, offline, battery powered metagenomics workflows. Meanwhile, MinION sequencing is becoming integral to our Arctic fieldwork programme, revealing glacier microbial biofilm community structures and metagenomes while deployed in field camps and stations. As the MinION platform, workflows and tools are maturing the prospect of characterising Earth’s microbiomes at source is starting to be realised.
Arwyn Edwards is a microbial ecologist at Aberystwyth University. He is currently a Senior Lecturer in Biology at the Institute of Biological, Environmental & Rural Sciences and Director of the Interdisciplinary Centre for Environmental Microbiology. His research group is focused upon the evolution, structure, function and stability of microbial communities in extreme environments in response to environmental changes at both societally-relevant and “deep” geo/evolutionary timescales. Arwyn particularly enjoys taking Nanopore sequencing to strange new places.Agenda
Wellcome Trust Sanger Institute
Kim graduated with a degree in Natural Sciences from the University of Cambridge, and joined Illumina as a research associate in sequencing R&D, where she worked on the MiSeq, NextSeq500 and Nextera. She moved to the Department of Medicine at Addenbrooke’s Hospital, Cambridge in 2012. Her PhD focused on using the Oxford Nanopore MinION for microbiological applications including detecting antimicrobial resistance genes and identifying plasmids. She joined the sequencing R&D team at Sanger in 2016 where she works with the MinION, GridION and PromethION.Agenda
University of California, San Fransisco
Metagenomic Nanopore Sequencing for Precision Diagnosis of Infectious Diseases
Metagenomic next-generation sequencing (mNGS) holds great promise for infectious disease diagnosis because of capacity to identify all potential pathogens – bacteria, viruses, fungi, and parasites – in a single assay. In June 2017, we completed the PDAID (“Precision Diagnosis of Acute Infectious Diseases”) study, a multi-hospital, nationwide prospective study of 220 patients, to evaluate the utility, outcome impact, and cost-effectiveness of a CLIA (Clinical Laboratory Improvement Amendments)-validated mNGS Illumina-based assay versus conventional microbiological testing for diagnosis of infectious causes of meningitis and encephalitis. We will discuss interim results from the PDAID study, and extension of mNGS to real-time metagenomic diagnosis of febrile illnesses in the field on a nanopore platform (the MinION sequencer from Oxford Nanopore Technologies). We will also discuss recent technological improvements that enable rapid and high-sensitivity detection (102 copies/mL) of pathogens from clinical body fluid samples, including the implementation of a “spiked primer” approach and the development of the SURPIrt real-time computational platform, which can analyze up to 1 million reads in <10 minutes on a laptop. Finally, we will describe ongoing efforts to clinically validate the nanopore platform in a CLIA laboratory, and to deploy this platform in remote field settings for infectious diseases testing, ranging from pandemic “hotspots” in Africa to outer space aboard the International Space Station (ISS).
Charles Chiu, M.D./Ph.D. is a microbiologist and infectious diseases physician who pioneered the development of metagenomic next-generation sequencing (mNGS) testing for clinical diagnosis of infectious diseases. He currently leads a translational research laboratory aimed at (1) sequencing-based clinical assay development and laboratory validation, (2) investigation of emerging pathogens (including Borrelia burgdorferi, Ebola virus, enterovirus D68, and Zika virus), (3) pathogen discovery in acute illness, and (4) new technology development such as nanopore sequencing. Dr. Chiu is the principal investigator of the 1-year (June 2016 – June 2017) nationwide, multi-hospital “Precision Diagnosis of Acute Infectious Diseases” study to evaluate the clinical impact and cost-effectiveness of a validated metagenomic next-generation sequencing assay for diagnosis of encephalitis and meningitis in hospitalized patients. He has authored more than 40 peer-reviewed publications since 2011, holds over 10 patents or patents, and serves on the scientific advisory board for Therabio, Inc. His translational work is supported by funding from the NIH, Abbott Laboratories, philanthropic organizations (Sandler, Bowes, Marcus, Charles and Helen Schwab, and Steve and Alexandra Cohen Foundations), and the California Initiative to Advance Precision Medicine.Agenda
Direct Determination of Mouse Genome-Wide, Allele-specific DNA Methylation from Nanopore Long-Read Sequencing.
Asymmetric expression patterns between the two parental alleles are critical for development of the mammalian embryo. This process known as imprinting involves differential DNA methylation of the parental genomes. We sequence mouse embryonic placenta tissue on the Oxford Nanopore MinION and exploit the long reads to determine, using novel and established methods, both haplotype and CpG methylation levels. Comparison with matched Reduced-Representation Bisulfite Sequencing data confirms the accuracy of the methylation calls, and highlights the improvement in haplotyping conferred by the longer reads. We successfully identify known imprinting control regions, as well as novel differentially methylated regions. Based on their proximity to hitherto unknown monoallelically expressed genes, we propose that some of these regions could constitute new imprinting control regions.
Scott Gigante is a first-year doctoral student in Computational Biology and Bioinformatics at Yale University. Under the guidance of Professor Hongyu Zhao, Scott is investigating immune cell quantification in cancer genomics. Prior to moving to Yale, Scott worked as a Research Assistant with Professor Terry Speed at the Walter & Eliza Hall Institute for Medical Research. Here, Scott began his research in nanopore sequencing, including the development of neural networks for basecalling and methylation detection as well as further downstream analysis of nanopore sequencing data. Scott completed a Bachelor of Science in 2015 as a Chancellor's Scholar and Australian Government New Colombo Plan Fellow at the University of Melbourne with a major in Mathematics & Statistics, specialising in Pure Mathematics.Agenda
Towards complete bacterial genomes from complex metagenomes using long-read sequencing
Microbial communities underpin all processes in the environment and have a direct impact on human health. However, despite their importance, only a tiny fraction of the millions of different microbes is known. This is mainly due to the immense difficulties of cultivating microbes from natural systems in the laboratory.The current standard approach to retrieve individual microbial genomes from complex samples is to use short-read sequencing (Illumina; approx. 2 x 125 bp) of multiple related samples combined with metagenomic differential coverage binning approaches. However, retrieving individual bacterial genomes from complex microbial communities is extremely challenging using short reads, due to the existence of multiple closely related strains. In this talk, I will share our experience in using high-throughput long-read data from Oxford Nanopore to recover individual genomes from complex metagenomes.
The overall aim of Mads’ research group is to enable investigation of unculturable microbes and microbial ecology in complex environments through the use of DNA sequencing. Topics including method development within genome-centric metagenomics, metatranscriptomics, and bioinformatics. A strong focus on new technologies related to novel applications, methods and software development.Agenda
Rijk Zwaan Breeding BV
Exploring new horizons in plantbreeding using Nanopore sequencing
In the plantbreeding industry collections of wild germplasm are essential for the introgression of new traits of agronomic importance into elite breeding material. A major challenge is the introgression of these traits into new varieties in a cost and time effective way. High quality de novo genome assemblies of diverse germplasms help to accelerate the molecular breeding process and enable faster introduction of new traits in the competitive vegetable seedmarket. Therefore Rijk Zwaan Breeding BV very recently set up Nanopore sequencing in an explorative set-up. Preliminary data will be shown.
In the In the past two decades Taco Jesse has built a track record in physical mapping, genome sequencing and map based cloning in the plant breeding industry. He started in the 1990's at KeyGene with BAC-based shotgun sequencing of Arabidopsis and participated in international projects sequencing tomato and potato. This work resulted in several co-publications in major journals and two patents. Since 2008 he is working at Rijk Zwaan Breeding BV in the Netherlands - a vegetable seed company - now as Team Coordinator NGS, exploring Nanopore technology in a highly motivated R&D driven plantbreeding environment.plantbreeding industry collections of wild germplasm are essential for the introgression of new traits of agronomic importance into elite breeding material. A major challenge is the introgression of these traits into new varieties in a cost and time effective way. High quality de novo genome assemblies of diverse germplasms help to accelerate the molecular breeding process and enable faster introduction of new traits in the competitive vegetable seedmarket. Therefore Rijk Zwaan Breeding BV very recently set up Nanopore sequencing in an explorative set-up. Preliminary data will be shown.Agenda
J. Craig Venter Institute
High molecular weight DNA enables ultra-long reads for genome assembly
High quality assemblies from larger complex genomes require reads that span repeat structures. Oxford Nanopore can generate ultra-long reads in excess of a megabase (Mb) to date and on average >50 kilobase (kb) if high molecular weight (HMW) DNA is used for library prep. These ultra-long reads enable assembly of complex nested transposable elements, large tandemly repeated gene clusters, ribosomal DNA, centromeres, engineered regions such as T-DNA insertions and complete sequencing of organelle genomes. However, isolating HMW DNA can be a challenge that requires special techniques specific to the organism and tissues being used for sequencing. I will discuss some of the techniques we have utilized to isolate HMW DNA and ultra-long reads such as in-gel extraction without DNA shearing for 1D library prep. Furthermore, I will provide some examples of the strengths and limitation of current ultra-long read lengths at resolving complex regions in the assembly of larger genomes.
Dr. Michael is Professor and Director of Informatics at the J. Craig Venter Institute (JCVI) in La Jolla, CA USA where his group focuses on reading and writing genomes. Before JCVI, Dr. Michael was Director of Genomics and Research Fellow at Abbott Labs where his team developed methods to edit the epigenome. Dr. Michael also led the Genome Center at Monsanto Company, where his team sequenced thousands of prokaryote and hundreds of eukaryotic genomes. Dr. Michael started his lab at the Waksman Institute and Rutgers University where he leveraged next generation sequencing to understand how the epigenome integrates environmental information. Dr. Michael received his PhD from Dartmouth College, BA from the University of Virginia, and conducted Postdoctoral research at The Salk Institute for Biological Studies.Agenda
Centers for Disease Control and Prevention, USA
Direct RNA sequencing of Influenza viral RNA using the MinION nanopore sequencer
The MinION from Oxford Nanopore Technologies (ONT) has the ability to sequence polyadenylated mRNA without cDNA synthesis or amplification. By modifying the reverse transcriptase adapter (RTA) from the ONT Direct RNA kit to hybridize to the universal 3’ end of the influenza viral genome, we have adapted this technique to target influenza viral RNA for direct RNA sequencing. Using this strategy on RNA from highly purified A/Puerto Rico/8/1934 (H1N1) virus particles, we sequenced viral RNA on the MinION and generated 99% accurate consensuses for all eight segments. Additionally, we were able to demonstrate efficient RNA sequencing of the influenza genome using a crude total RNA preparation from the allantoic fluid of embryonic chicken eggs inoculated with the same influenza virus. Based on our results, we postulate a similar modification strategy can be used to target and sequence all influenza virus RNA species during an active influenza infection.
Dr. Matthew Keller is an ORISE postdoctoral fellow under the mentorship of Dr. John Barnes of the Influenza Genomics Team at the Virology, Surveillance, and Diagnosis Branch of the CDC’s Influenza Division. Dr. Keller received his PhD in Biochemistry and Molecular Biology from the University of Georgia where he studied biofuel production in thermophiles and authored 10 publications. Dr. Keller joined the CDC in April of 2017 where he is currently investigating nanopore sequencing for the surveillance and study of influenza viruses.Agenda
Garvan Institute for Medical Research
ONT offers more than ultra long reads and the capacity to detect base modifications in real-time. I will elaborate on the real-time nature of nanopore sequencing, where biopolymer subsequences can be interrogated directly as they transit through the pore. To achieve this, it is essential to venture into ‘squiggle space’—the raw or normalised signal data produced by Oxford Nanopore flowcells. I will describe a method to de-multiplex barcodes in squiggle space, using supervised and unsupervised approaches. So far, this approach performs well on standard ONT barcodes, and more than doubles the amount of correctly demultiplexed barcodes from single cell RNAseq data when compared to standard barcode matching strategies. Furthermore, I will demonstrate how this approach can be used to improve the assembly of full-length cDNAs from thousands of cells.
Martin Smith is currently the head of the Genomic Technologies program at The Kinghorn Centre for Clinical Genomics, part of the Garvan Institute of Medical Research in Sydney, Australia. His group works closely with the newly established Garvan-Weizmann Centre for Cellular Genomics. Martin completed his PhD in genomics and computational biology in 2012 at the Institite for Molecular Bioscience, University of Queensland. He is originally from Montreal, where he studied Bioinformatics (MSc) and Microbiology (BSc).Agenda
The Jackson Laboratory
Nanopore Sequencing Reveals High-Resolution Structural Variation in the Cancer Genome Abstract Acquired genomic structural variants (SVs) are major hallmarks of the cancer genome. Their complexity has been challenging to reconstruct from short-read sequencing data. Here, we exploit the long-read sequencing capability of the nanopore platform using our customized pipeline, Picky, to reveal SVs of diverse architecture in a breast cancer model. From modest sequencing coverage, we identified the full spectrum of SVs with superior specificity and sensitivity relative to short-read analyses and uncovered repetitive DNA as the major source of variation. Examination of the genome-wide breakpoints at nucleotide-resolution uncovered micro-insertions as the common structural features associated with SVs. Breakpoint density across the genome is associated with propensity for inter-chromosomal connectivity and transcriptional regulation. Furthermore, an over-representation of reciprocal translocations from chromosomal double-crossovers was observed through phased SVs. The comprehensive characterization of SVs using the robust long-read sequencing approach in cancer cohorts will facilitate strategies to monitor genome stability during tumor evolution and improve therapeutic intervention. Biography Dr. Wei joined the Jackson Laboratory in 2016 as the Director of Genomic Technologies, where she actively engages the development and application of innovative sequencing based genomic technologies to explore genome function and make genome-based medical discoveries for solutions in treating human disease. Dr. Wei got her Ph.D. (Microbiology) from University of California at Davis. Prior to that she was at the Joint Genome Institute (JGI), joining in 2010. She was responsible for Department of Energy (DOE) user facility genomic sequencing operation as well as sequencing related technology development effort within the Genomic Technology Department. Dr. Wei moved to JGI after an eight-year stint at Genome Institute of Singapore (GIS) and was responsible for establishing a sequencing technology platform and genome biology program. Dr. Wei group’s research activity involves the development of innovative sequencing-based genomic technologies to explore functional genomic elements for transcriptional and epigenetic regulation. She applies these approaches to interrogate genomic, characterize transcriptomes and chromatin structures.Agenda
Wellcome Trust Centre for Human Genetics
Nanopore cDNA sequencing produces complex transcriptomes for sensitive and accurate gene expression profiling
In the last decade, transcriptome sequencing with Illumina (RNAseq) has contributed tremendously to high-impact research providing far-reaching insights into the complexity of the expressed genome. This year at Oxford Genomics Centre (OGC) ~75% of our Illumina sequencing, amounting to over 30TB of data, has been performed on RNA libraries, highlighting the prevalence of RNAseq in genomics. Standard Illumina RNAseq, although the current gold-standard in gene expression profiling, sequences short RNA fragments and therefore has inherent limitations and biases. Recently, Oxford Nanopore Technologies (ONT) launched kits for transcriptome profiling with full-length cDNA transcripts. We evaluate ONT cDNA sequencing by analysing Universal Human RNA Reference (UHRR) libraries with different combinations of ERCC and SIRVs spike-ins and further compare them against Illumina libraries. We observe comparable expression level and fold change quantification in both library types. Despite low sequencing depths (~5million reads), ONT cDNA libraries have high complexity and detect a slightly different pool of transcripts compared to Illumina. These preliminary findings suggest high potential for nanopore cDNA libraries for accurate gene-expression quantification and identification of isoforms even at low sequencing depths.Agenda
Johns Hopkins University
Bacterial DNA modifications with Nanopore Sequencing
Nanopore sequencing has enormous potential in directly detecting covalent modifications and non-canonical bases in DNA; unlike traditional sequencing-by-synthesis technologies, it can distinguish covalently modified nucleotides directly through their modulation of the electrolytic current. We can take advantage of the long read lengths (>10kb) generated by nanopore sequencing to precisely call modified base patterns. We have already demonstrated the accuracy and some of the promise of nanopore sequencing in calling 5-methylcytosine in a CG context using a hidden Markov model. We have now trained our model on other modified (non-canonical) nucleotides, including 5-methylcytosine in a non-CG context, N6-methyladenosine, 4-methylcytosine, and deoxyuracil. We have estimated the efficiency of de novo identification of the methylation motifs and trained calling of these modifications in bacterial genomic and plasmid samples.
Yunfan Fan is a PhD student in the department of Biomedical Engineering at Johns Hopkins University, where she also earned undergraduate degrees in Biomedical Engineering and Electrical Engineering. She works in Dr. Winston Timp’s lab, focusing on projects involving infectious disease sequencing and epigenetics.Agenda
Understanding phage-bacterial host interactions for smarter therapeutics
EpiBiome is working on bacteriophage therapeutics to meet the growing threat of antibiotic resistance. Bacteriophage are viruses that infect a narrow range of bacteria and can be used as a precisely targeted therapeutic. In order to effectively design phage cocktails, it is important to study both the phage’s and the bacterial host’s genomes. By incorporating the MinION into our current sequencing pipeline, we can now complete the genomes of ETEC bacteria in order to understand resistance pathways used by the bacteria to avoid phage infection.
Anika Kinkhabwala received her Ph.D. from Stanford University in the laboratory of Nobel Laureate Prof. W. E. Moerner. She currently leads the genomics team at EpiBiome, Inc., a precision microbiome engineering company working to combat infectious diseases using bacteriophage therapy. Her group focuses on understanding the interaction between phages and their bacterial hosts in order to better design bacteriophage cocktails.Agenda
Dalhousie University, Canada
Jon Jerlström Hultqvist
Nanopore sequencing for genomics of heterotrophic protists
To date, the main focus of the sequencing revolution has been on a limited set of eukaryotes, mostly consisting of animals, plants, fungi and their parasites. However, the vast majority of eukaryotic diversity is microbial and free-living and to fully appreciate the path to parasitism we need to sample the genomes of these organisms. We seek to obtain genome information from a diverse set of poorly sampled organism groups, some of which are related to significant parasites in humans and animals. To this end, we have established nanopore sequencing as powerful tool to obtain near-complete genome sequences of a varied set of microbial eukaryotes grown in prokaryotic co-cultures. Long-read sequence assemblies in combination with polyA-selected RNAseq data have proven to be a powerful tool to enable accurate taxonomic binning of eukaryotic genomes from complex starting cultures.
After completing a Master Science in Biotechnology at Uppsala University I joined the lab of Prof. Staffan Svärd at Uppsala University in Sweden. In 2012 I earned a Ph.D. in Microbiology on genomics, transcriptomics and cell biology of Diplomonads (Spironucleus and Giardia). The following year I started a Post Doc in the group of Prof. Dan Andersson at Uppsala University on experimental approaches to investigate the impact of lateral gene transfer in antibiotic resistance and metabolism in E. coli. I joined the Roger Lab at Dalhousie University, in Halifax in September 2016 for my second Post Doc, using genomics to study the adaptation to low oxygen conditions in protists.Agenda
Talks from Scientists using nanopore sequencing in the auditorium.View agenda
Choose from 3 breakout sessions per day. Topics will include RNA/cDNA, Microbiology and Data analysis amongst others.
Over 30 posters were submitted last year. A selection of these are invited to present a Lightning talk in the main auditorium.
Product demo area
An opportunity to see the latest version of our products in action. You can also take part in MinKNOW, Albacore and flow cell loading clinics.
Dinner and open bar; this will take place on Thursday 30th November and is included in the price of your ticket.
1-day workshop including hands-on practical which will take you through sample preparation, running the device to data analysis. Available for Nanopore Community members only. Suitable for beginner to intermediate nanopore users.
|Hotel||Walk from venue||Standard rate|
|Hilton Garden Inn||9 min drive / 6 min train / 20 min walk||$219|
|Paramount NYC||17 min drive / 15 min train / 30 min walk||$279|
|Holiday Inn||10 min drive / 9 min walk||$309|
|Radisson Hotel Martinique||12 min drive / 15 min walk||$325|
|Hyatt Herald Square||10 min drive / 12 min train / 15 min walk||$349|
|Doubletree by Hilton Hotel New York City- Chelsea||7 min drive / 12 min walk||Best Available Rates|
|Hilton Fashion District||11 min drive / 10 min walk||Best Available Rates|