An International Symposium of the Association of Biomolecular Resource Facilities

Genomics Oral Presentations

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Session Type: Workshop

  • Complementary approaches to profiling nascent protein synthesis in peripheral neurons in vivo and in vitro
    Zachary Campbell UT-Dallas Genome Center And The Dept. Of Biological Sciences
    Translational control is a dominant theme in neuronal plasticity. Messenger RNA (mRNA) is subject to dynamic regulation by multi-protein regulatory complexes. These large assemblies enable signal-dependent control of protein synthesis. Tremendous progress has been made on the proximal signaling events that control translation regulation in the nervous system leading to neuronal plasticity (e.g. learning and memory, LTP/LTD, neurodevelopmental disorders, and many forms of chronic pain). However, astonishingly little is known about the downstream mRNA targets that are translated to produce the new proteins that mediate this plasticity. We are establishing a novel resource that comprehensively captures nascent protein synthesis levels in sensory neurons called nociceptors using next-generation sequencing to profile translation. Chronic pain is characterized by persistent plasticity in nociceptors and is a devastating condition with a lifetime incidence greater than 33%. Poorly managed pain creates an enormous burden on our healthcare system and produces tremendous human suffering. This resource provides insight into how pain evoking stimuli trigger dynamic alterations in the landscape of protein synthesis thereby facilitating nociceptor plasticity. The data have clear implications for improved pain treatment and will serve as a paradigm for understanding neuronal plasticity in other areas of neuroscience.
    Session Type: Workshop
  • 3D-Printed Continuous Flow PCR Microfluidic Device for Field Monitoring of Bacterial DNA
    Elizabeth Hénaff Weill Cornell Medicine
    Environmental metagenomics – measuring bacterial species and gene content from environmental samples – is relevant in many contexts, including agriculture, land stewardship, and disease outbreak monitoring. Indeed, soil bacteria have been shown to influence crop outcome, the response to harmful algal blooms is largely dependent on response time, and pathogen mapping is relevant on the urban scale. Many of these issues are of direct concern to the public, and citizen scientists are increasingly becoming part of such studies, enabling data collection at an unprecedented scale. Furthermore, engaging citizens to monitor their environment has a number of positive impacts on the public perception of human and environmental safety. Indeed, recruiting non-scientists for sample collection has enabled projects such as Ocean Sampling Day (OSD; https://www.microb3.eu/myosd/how-join-myosd), the PathoMap study (http://www.pathomap.org), and the MetaSUB consortium (http://metasub.org) by recruiting numbers of volunteers across the world. The sense of agency derived from the ability to personally monitor one’s environment enables data-driven discussions around the microbial milieu and can help alleviate, or justify, people’s concerns around the enforcement of best environmental practices. While high-throughput whole-genome sequencing, as used by PathoMap and MetaSub, provides an in-depth view of environmental metagenomes and is necessary for full functional characterization, often the detection of indicator species or genes can be sufficient to engage in first response. Thus detection of specific DNA markers, for microbial species or functional plasmid identification, would be relevant for context-specific data collection. Here we describe a cheap, scalable and easy-to-use 3D printed device which implements continuous flow PCR for specific detection of DNA markers, and is robust enough that it may be used by non-scientists. The hardware plans (3D print files and circuit designs) will be made available under the Open Source Hardware Association (http://www.oshwa.org/) so that they may be replicated and implemented freely.
    Session Type: Workshop
  • An automated low-volume, high-throughput library prep for studying bacterial genomes
    Jon Penterman MIT
    In depth, genomic studies on bacterial isolates from clinical or environmental settings can be prohibitively expensive. The most significant expense for such studies is the preparation of sequencing libraries. Sequencing libraries can be prepared by ligating adaptors to end-repaired DNA (many kits) or by tagmentation and PCR enrichment (Illumina NexteraXT). For large-scale studies, the NexteraXT kit is a popular choice because the two-step, single tube protocol uses unprocessed gDNA as the input (versus fragmented gDNA for non-tagmentation protocols). Here we describe a high throughput, low volume NexteraXT protocol that significantly lowers library preparation costs. Central to this protocol is the Mosquito HTS robot, a small-volume liquid handler that aspirates and dispenses on a 96 or 384-well scale. We miniaturized the NexteraXT reaction to 1/12th the normal scale on the Mosquito and took advantage of a preexisting Tecan Evo robot to completely automate the remaining parts of the library prep service (normalization of DNA input for prep, library normalization, pooling). The sample dropout rate for this protocol is low, and overall sequencing coverage of both control and experimental samples is similar to that seen in NexteraXT libraries prepped at the normal scale. By reducing reagent usage and labor input on a per sample basis, we have made large bacterial gDNA sequencing projects more financially feasible.
    Session Type: Workshop
  • MetaSUB: Metagenomics Across the World's Cities
    Ebrahim Afshinnekoo Weill Cornell Medicine/New York Medical College
    The Metagenomics and Metadesign of the Subways and Urban Biomes (MetaSUB) International consortium is a novel, interdisciplinary initiative made up of experts across many fields including genomics, data analysis, engineering, public health, and design. Just as there is a standard of measurement of temperature, air pressure, wind currents– all of which are considered in the design of the built environment– the microbial ecosystem is just as dynamic. Thus, it should also be integrated into the design of cities. By developing and testing standards for the field and optimizing methods for urban sample collection, DNA/RNA isolation, taxa characterization, and data visualization, the MetaSUB International Consortium is pioneering an unprecedented study of urban mass-transit systems and cities around the world. These data will benefit city planners, public health officials, and designers, as well as discovery new species, biological systems, and biosynthetic gene clusters, thus enabling an era of more quantified, responsive, and “smarter cities.” During this talk we’ll share preliminary results from pilot studies carried out across the cities in the consortium including taxa classification, functional analysis, and antimicrobial resistance markers.
    Session Type: Workshop
  • Precision Metagenomics: Rapid Metagenomic Analyses for Infectious Disease Diagnostics and Public Health Surveillance
    Ebrahim Afshinnekoo Weill Cornell Medicine/New York Medical College
    Next-generation sequencing technologies have ushered in the era of Precision Medicine, transforming the way we diagnose and treat cancer patients. Subsequently, the advent of these technologies has created a surge of microbiome and metagenomics studies over the last decade, many of which are intent upon investigating the host-gene-microbial interactions responsible for the development of chronic disorders. As we continue to discover more information about the etiology of complex chronic diseases associated with the human microbiome, the translational potential of metagenomics methods for the treatment and rapid diagnosis of infectious diseases is also becoming abundantly clear. Here, we present a robust protocol for the utilization and implementation of “precision metagenomics” across various platforms on clinical samples. Such a pipeline integrates DNA/RNA extraction, library preparation, sequencing, and bioinformatics analysis for taxa classification, antimicrobial resistance marker screening, and functional analysis. Moreover, the pipeline is built towards three tracks: STAT for rapid 24-hour processing as well as Comprehensive and Targeted tracks that take 5-7 days for less urgent samples. We present some pilot data demonstrating the applicability of these methods on cultured isolates and finally, we discuss the challenges that need to be addressed for its full integration in the clinical setting.
    Session Type: Workshop

Session Type: Research Group

  • The eXtreme Microbiome Project “Down Under”: Metagenomic Adventures in the Southern Hemisphere.
    Ken McGrath Australian Genome Research Facility
    The eXtreme Microbiome Project (XMP) is a global scientific collaboration to characterize, discover, and develop new pipelines and protocols for extremophiles and novel organisms from a range of extreme environments. Two of the recent study locations are in the southern hemisphere: Lake Hillier, a bright pink hypersaline lake located on the Recherche Archipelago in Western Australia; and Lake Fryxell, a permanently frozen freshwater lake located in the Dry Valleys of Antarctica. Sampling in such remote environments comes with inherent challenges for sample collection and preservation, ranging from shark bite to frostbite. Despite these challenges, the XMP team has recovered samples of these microbial communities, and have analysed the metagenomes using a range of sequencing platforms, revealing the composition of the microbial communities that inhabit these extreme environments of our planet. Our results identify an abundance of highly specialised organisms that thrive in these locations, as well as demonstrate the utility of third-generation sequencing platforms for in situ analysis of microbial communities.
    Session Type: Research Group
  • Metagenomic Analysis using the MinION Nanopore Sequencer
    Ken McGrath Australian Genome Research Facility
    Metagenomic DNA from microbial communities can be recovered from virtually anywhere on our planet, yet determining the composition of those communities remains a challenge, in part due to bioinformatics complexity of assembling sequencing reads from a cocktail of similar genomes. We compare the Oxford Nanopore MinION platform to other sequencing platforms and demonstrate that the longer reads from the MinION can compensate for the higher error rates, resulting in comparable metagenomics profiles. The reduced sample processing time and the enhance portability of the MinION make this platform a useful tool for studying microbial communities, particularly those in remote or extreme environments.
    Session Type: Research Group
  • Cross-Site Comparison of Ribosomal Depletion Kits for Illumina RNAseq Library Construction
    Stuart Levine MIT
    Ribosomal RNA (rRNA) comprises at least 90% of total RNA extracted from mammalian tissue or cell line samples. Informative transcriptional profiling using massively parallel RNA sequencing technologies requires either enrichment of mature poly-adenylated transcripts or targeted depletion of the rRNA fraction. The latter method is of particular interest because it is compatible with degraded samples such as those extracted from FFPE, and it also captures transcripts that are not poly-adenylated such as some non-coding RNAs. Here we provide a cross-site study that evaluates the performance of ribosomal RNA removal kits from Illumina, Takara/Clontech, Kapa Biosystems, Lexogen, New England Biolabs and Qiagen on intact and degraded RNA samples. We find that all of the kits are capable of performing significant ribosomal depletion, though there are large differences in their ease of use. Most kits perform well on both intact and degraded samples and all identify ~14,000 protein coding genes from the Universal Human Reference RNA sample at >1FPKM, though the fraction of reads that are protein coding or in annotated lncRNAs varies between the different methodologies. These results provide a roadmap for labs on the strengths of each of these methods and how best to utilize them.
    Session Type: Research Group
  • Genomics Research Group 2016-17 study: A multiplatform evaluation of single-cell RNA-seq methods.
    Sridar Chittur University At Albany, SUNY
    The Genomics Research Group (GRG) presentation will describe the current activities of the group in applying the latest tools and technologies for single cell transcriptome analysis to determine the advantages and disadvantages of each of the platforms. This project involves the comparison of gene expression profiles of individual SUM149PT cells treated with the histone deacetylase inhibitor TSA vs. untreated controls. The goals of this project are to demonstrate RNA sequencing (RNA-seq) methods for profiling the ultra-low amounts of RNA present in individual cells, and to demonstrate the use of the various systems for cell capture and RNA amplification including Fluidigm, Wafergen, fluorescence activated cell sorting (FACS), 10x Genomics, and Illumina’s joint venture with BioRad on the ddSEQ platform. In this session, we will discuss the technical challenges, results from each of these projects, and some key experimental considerations that will help leverage optimal results from each of these technologies.
    Session Type: Research Group

Session Type: Scientific Session

  • Offering Single-Cell RNA-Seq as a Core Service
    Anoja Perera Stowers Institute For Medical Research
    High-throughput transcriptome analysis of single cells enables gene expression measurement of individual cells and allows the discovery of heterogeneity within a given cell population. On the wet lab side, meeting the increasing demand for single-cell data can be challenging due to many factors, including cost of instrumentation, library construction, and labor requirements. Since this field is growing very rapidly, it also becomes a challenge to evaluate new instrumentation and make appropriate investments. Recently, we have explored several methods to address these challenges. Our initial efforts focused on cost reductions by reducing reaction volumes using small volume pipetting robots. Here, we looked at both the Formulatrix’s Mantis and TTP Labtech’s Mosquito. As more advanced single-cell instrumentation came on the market, we evaluated WaferGen’s ICELL8 and the 10X Genomics’ Chromium Single Cell System. These assessments have led to two options for single-cell RNA-Seq at our core. Using the 10X Genomics’ Chromium System, we are able to rapidly evaluate thousands of individual cells at a greatly reduced cost. Our second workflow involves setting up quarter-sized reactions using the Formulatrix’s Mantis robot. This method is used in studies where we want to further evaluate a selected population of cells as well as on rare cell populations. At the Stowers Institute for Medical Research, where the Molecular Biology Core Facility is a medium-sized operation, these two workflows allow us to meet the current single-cell transcriptomics needs.
    Session Type: Scientific Session
  • Only the shallow know themselves: Deep sequencing of Single Cell RNA
    Seth Crosby Washington University
    Current methods for quantifying molecular states of cells often depend on estimating the mean gene expression values from hundreds to millions of cells. Given the heterogeneity of cell populations, these mean values miss differences and, perhaps, interactions within a cell population. RNA with low copy number, which may exert important functions, is usually undetectable or regarded as noise in bulk cell-averaging methods. By combing a variety of small-volume library prep methods and massively parallel next generation sequencing (NGS), single-cell RNA sequencing (scRNA-seq) provides RNA expression profiles of individual cells. As a result, scRNA-seq can identify rare cell types within a cell population, creating and tracking subpopulation structures. Uncommon transcripts, which are obscured in bulk sequencing can be revealed. Even genetically identical cells, under the same environment, can display variability of gene and protein expression levels. I will discuss our experience with various scRNA platforms, the value of very deep sequencing of properly prepared scRNA libraries, and the importance of applying proper informatics tools to the various types (wide/shallow vs narrow/deep) of scRNA-seq data.
    Session Type: Scientific Session
  • Perspectives for Whole Cell Microbial Reference Materials
    J. Russ Carmical Baylor College Of Medicine
    With the exception of the Microbiome Quality Control (MBQC), very little has been published on best practices and reference standards for microbiome and metagenomic studies. As evidenced by recent publication trends, researchers are moving the field toward commercial development at a rapid pace.  If these analyses are to ever evolve into reliable assays (e.g. for clinical diagnostics), the measurement process must be regularly assessed to ensure measurement quality.  A key aspect of this validation is the routine analysis of reference materials as positive controls. A reference material (RM) is any stable, abundant, and well characterized specimen that is used to assess the quantitative and/or qualitative validity of a measurement process. The focus will be on the use of whole cell microbial reference materials to characterize metagenomic analyses from start to finish. We’ll discuss 3 key categories (environmental samples, in vitro models for microbial ecosystems, pure microbial isolates) of reference standards and the challenges in characterizing those standards.
    Session Type: Scientific Session
  • Current Innovations for Metagenomics used in Antarctica
    Scott Tighe Uiversity Of Vermont
    Novel advancements in genomics by the metagenomics research group have made it possible to extract, isolate, and sequence DNA recovered from ancient microbial biofilms from buried paleomats in Antarctica. These techniques include high performance DNA extraction protocols using the multi-enzyme cocktail branded as MetaPolyZyme combined with a new hybrid DNA extraction mag-bead kit. These techniques allow for the recovery of high molecular weight DNA suitable for Oxford Nanopore sequencing both in the Crary lab in McMurdo Station and while in the field in Antarctica as well as high resolution sequencing using the PacBio and Illumina systems.
    Session Type: Scientific Session
  • Clinical Whole Genome Sequencing: Challenges, Opportunities and Insights from the First Thousand Genomes Sequenced
    Shawn Levy HudsonAlpha Institute For Biotechnology
    Major technological and computing advancements have allowed routine generation of whole genome sequence data on hundreds of thousands of people over the last several years. As the ability to analyze and annotate genomes has improved, the clinical utility and impact has greatly enhanced the ability to discover and define the genetic causes of a wide variety of human phenotypes. In December of 2015 we launched a clinical laboratory focusing on whole-genome sequencing and we have used that infrastructure to sequence over 1,000 genomes for translational and clinical projects, delivering results back to patients and secondary findings to parents. During the course of these studies, we have learned a number of valuable lessons and have more appropriately calibrated our expectations and uses for whole genome sequencing. This presentation will highlight those successes and challenges and discuss the dynamic and powerful capabilities of genomics for both routine clinical use as well as in the treatment of critically ill patients.
    Session Type: Scientific Session
  • A profusion of confusion in genomic methods
    James Hadfield CRUK Cambridge Institute
    The number of next-generation sequencing (NGS) methods has grown to almost 400 in the past ten years. Each method includes specific processing steps that adapt NGS to address an expanding range a genomic applications allowing researchers to ask varied biological questions - but only if they know the method exists. Most methods are given names by their creators but even the most commonly used method “RNA-seq” is often used to refer to very different methodological approaches or biological applications. Method naming has not been controlled, but organising methods and structuring naming to allow new users to navigate the NGS publication landscape is overdue. I will present a few of the more confusing naming examples to highlight the problem, and describe a definitive list of methods, ordered by function, made available and maintained on a community wiki.
    Session Type: Scientific Session
  • Genome in a Bottle: So you've sequenced a genome, how well did you do?
    Justin Zook National Institute Of Standards And Technology
    Six new extensively characterized whole genome reference samples were released by the National Institute of Standards and Technology with the Genome in a Bottle Consortium in September 2016. These samples come from an Ashkenazim trio and a Chinese trio in the Personal Genome Project (PGP). Four of these are available from large batches of cell lines as NIST Reference Materials (RMs), in addition to the pilot GIAB genome (NA12878) Since the release of the pilot genome, we have developed the methods to form high-confidence variant calls from multiple datasets into a robust, reproducible process that were applied to five GIAB genomes for GRCh37 and GRCh38. The new high-confidence calls cover 88-90% of the genome, in comparison to ~78% of the genome covered by our previous version of calls from 2015. These data have been used extensively; e.g., the GIAB ftp at NCBI has had ~50,000 downloads from ~1000 unique IPs per month in 2016. We have also worked with the Global Alliance for Genomics and Health Benchmarking Team to develop standardized performance metrics and tools to compare variant calls to our benchmark calls. GIAB is currently integrating short, long, and linked read data to form high-confidence characterization of more difficult variants and difficult regions of the genome. GIAB is also exploring samples of additional ancestries and cancer samples for reference material development.
    Session Type: Scientific Session

Session Type: Platinum Presentation

  • Accelerating Research with 3D Biology™: Simultaneous Single-molecule Quantification of DNA, RNA, and Protein Using Molecular Barcodes.
    Niro Ramachandran NanoString Technologies
    NanoString is pioneering the field of 3D Biology™ technology to accelerate the rate of research and maximize the amount of information that can be generated from a given sample. 3D Biology is the ability to analyze combinations of DNA (detect SNVs and InDels), RNA (Gene Expression or Fusion transcript detection), and Protein (abundance and post- translational modifications) simultaneously on a NanoString nCounter® system. We will highlight the use of SNV Technology in detecting cancer driver mutations, the utility of multiplexed DNA-labeled antibody approaches to quantify protein expression levels from small amounts of sample (both lysate and FFPE), and demonstrate the utility of multi-analyte analysis and the novel insights this approach can uncover.
    Session Type: Platinum Presentation
  • Automating CRISPR mutation detection and zygosity determination
    Kyle Luttgeharm Advanced Analytical Technologies
    While CRISPR gene editing is rapidly advancing becoming more economical and efficient, Protocols to identify CRISPR mutations and determine the zygosity of these mutations are time consuming and often involve costly sequencing steps. To overcome this limitation, many researchers are turning to heteroduplexing and enzymatic mismatch cleavage assays to rapidly screen for mutated lines. Despite this growing interest, few studies to optimize heteroduplex cleavage assays in relation to different mutations and lengths of PCR products have been performed. Additionally, sequencing has continued to be required for zygosity determination of individual diploid cell lines/organisms. Using Advanced Analytical Technologies Inc. Fragment Analyzer™ Automated Capillary Electrophoresis System and synthetic genes to mimic different CRISPR mutations, we developed an optimized heteroduplex cleavage assay employing T7 Endonuclease I to detect a wide variety of common CRISPR mutations including both insertion/deletions and single nucleotide polymorphisms. In order to decrease the number of individual cell lines sequenced we developed statistical models that relate heteroduplex formation to the number of mutated alleles in individual diploid cell lines/organisms. This protocol allows for accurate prediction of monoallelic, diallelic homozygous, and diallelic heterozygous events. Having a single high-throughput protocol that results in cleavage of multiple types of mutations while also determining the number of mutated alleles would allow for efficient screening of CRISPR mutant populations at a level currently not feasible.
    Session Type: Platinum Presentation
  • Advancements in NGS sample preparation for low input and single cells
    Andrew Farmer Takara Bio USA Inc
    Experimental approaches involving RNA-seq and DNA-seq have led to significant advancements in fields such as developmental biology and neuroscience, and are increasingly being applied towards the development of diagnosis and novel treatments for human disease. Our SMARTer NGS portfolio for DNA and RNA sequencing enables generation of libraries from single cell, degraded, and other low-input sample types . Together our products cater not only to generating sequencing libraries from difficult to obtain samples, but also to all major applications ( Differential gene expression analyses, Immune-profiling, Epigenomic profiling, Target enrichment, mutation detection for low frequency alleles and copy number variation). SMARTer methods perform consistently across a range of sample types and experimental applications, and are capable of processing low-input sample amounts. In this talk, we will present recent developments to the DNA and RNA seq portfolio.
    Session Type: Platinum Presentation

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