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2015 ABRF Metagenomics Research Group

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2015 ABRF Metagenomics Research Group 2015 ABRF Metagenomics Research Group The Extreme Microbiome Project (XMP) and Development of New Standards for Metagenomics and Microbiome Applications Scott Tighe University of Vermont Overview • About the MGRG • Mission Statement • Membership • Collaborators and Contributors • Current Projects • Development of Microbial Reference Standards • Development of a multi-lytic enzyme mix (with Sigma) • Development and evaluation of metagenomics preservatives • The Extreme Microbiome Study (XMP) • Study Sites • Methods • Bioinformatics • Data About the MGRG • New research group proposed and approved in March 2014 • Started recruiting members at the general meeting 2014 in Albq. • First was Kelley Thomas • Includes an Analytics group, Bioinformatics, and “Earth sciences” • Microbiologists, geneticists, computational biologist, geochemists, oceanographers, ecologists. • Focus on developing reference standards and reagents which are currently lacking in microbiome studies. • Development of standardized techniques and protocols • Working with corporates partners for developing new kits and reagents • Study extreme and unique environments with whole genome shotgun sequencing for metagenomics and metatranscriptomic approaches. Mission Statement The goals of the Metagenomic Research Group is to evaluate, study, and refine methodologies related to any aspect of metagenomics and microbiome studies. This includes study designs, controls, detection methods, and bioinformatics pipelines and software. The overall intent of ABRF MGRG is to assist in the advancement of the science of Metagenomics with the result of generating more accurate results and standard methods. Membership • Scott Tighe (Chair) University of Vermont • Ebrahim Afshinnekoo Weill Cornell Medical College • Nadim Ajami Baylor University • Don Baldwin MicroPath ID Diagnostics • Nathan Bivens University of Missouri • Russ Carmical UTMB - Galveston • Stefan J Green University of Illinois at Chicago • Samantha Joye University of Georgia • Jodie Lee American Type Culture Collection (ATCC) • Shawn Levy HudsonAlpha Institute for Biotechnology • Christopher Mason Weill Cornell Medical College • Ken McGrath Australian Genome Research Facility • Natalia G. Reyero Vinas MIssissippi State University • Matthew L Settles University of Idaho • Kelley Thomas University of New Hampshire • Noah Alexander Weill Cornell • Tim C Hunter (ABRF EB Liaison) University of Vermont Collaborators and Contributors • Diana Krawczyk Greenland Institute of Natural Resources • Jill Mikucki University of Tennessee • Svein-Ole Mikalsen University of Faroe Islands • John M Lizamore Dept of Parks and Wildlife Don Cater Western Australia Government Industry Partners and Contributors • Craig Rowell Illumina Corp Special Projects Coordinator • Aaron Sin Sigma Chemical Bob Gates • Liz Kerrigan American Type Culture Collection Jodie Lee • Carlos Merino DNA Genotek Aaron Del Duca • Nezar Rghei Norgen Biotek Corp. What are the Challenges with Metagenomics and where can the MGRG help ? DNA Extraction Efficiency DNA extraction efficiency is highly variable . Believe it or not, a bacteria or fungus that you can grow, might not release any DNA or RNA and will be missed . No Standard method . Different techniques have varying yields . No standard Enzyme Mix…MACL4 ABRF Nucleic Acids Research Group 2012-2013 Study Evaluating DNA Extraction Methods for Metagenomic Analysis V. Nadella1, J. Holbrook2, R. Carmical3, M. Robinson4, C. Rosato5, H. Auer6, N. Beckloff7, Z. Herbert8, S. Chittur9, A. Perera10 , W. Trimble11, S. Tighe12 1Ohio University, 2Nemours/A.I. DuPont Hospital for Children, 3University of Texas Medical Branch, 4 University of Zurich, Switzerland, 5Oregon State University, 6Institute for Research in Biomedicine, Barcellona, Spain, 7Case Western Reserve University, 8Dana Farber Cancer Institute, 9University at Albany-SUNY, 10 Stowers Institute for Medical Research, 11Argonne National Laboratory, 12University of Vermont. Current Challenges with Metagenomics . Library Amplification . GC bias . Sample loss due to clean up . Amplifying targets genes . 16s rDNA, ITS, or gyrase B (gyrB) . Primer Non-coverage can bias . ANAF- Aseptic Nucleic Acid Free . Filter Funnels . Bottles . Lack of Preservatives Coverage evaluation of universal bacterial primers using the . Software metagenomic datasets Dan-Ping Mao, Quan Zhou, Chong-Yu Chen and Zhe-Xue Quan BMC Microbiology 2012, 12:66 Reference Standards are rare as Hen’s Teeth • Human Microbiome project control- BEI-ATCC • http://www.beiresources.org/Catalog.aspx?q=HM-276D [or HM-782D] • Mock gDNA communities with even amounts 0.08 ng/μL of Acinetobacter baumannii§ 0.10 ng/μL of Actinomyces odontolyticus§ 0.04 ng/μL of Bacillus cereus§ 0.08 ng/μL of Bacteriodes vulgatus§ 0.04 ng/μL of Clostridium beijerinckii‡ 0.10 ng/μL of Deinococcus radiodurans§ 0.07 ng/μL of Enterococcus faecalis§ 0.09 ng/μL of Escherichia coli£ 0.03 ng/μL of Helicobacter pylori† 0.05 ng/μL of Lactobacillus gasseri‡ 0.06 ng/μL of Listeria monocytogenes§ 0.09 ng/μL of Neisseria meningitidis† 0.16 ng/μL of Propionibacterium acnes§ 0.14 ng/μL of Pseudomonas aeruginosa£ 0.06 ng/μL of Rhodobacter sphaeroides£ 0.05 ng/μL of Staphylococcus aureus§ 0.03 ng/μL of Staphylococcus epidermidis§ 0.04 ng/μL of Streptococcus agalactiae§ 0.06 ng/μL of Streptococcus mutans§ 0.04 ng/μL of Streptococcus pneumoniae§ Microbial Reference Standards • Initially developed by Nucleic Acid Research group • Arrangements between ATCC and ABRF-MGRG • gDNA and Whole cell microbial reference Standard • gDNA with known number of genomic copies –dPCR • Whole Cell with known concentrations of each species • Initially Class I genomes only. Classes II and III will be assembled later • Additional standards will include Eukaryotic Class I: Contains few repetitive sequences except for the ribosomal operons (5-7 kbp); can be reliably sequenced using short reads Class II: Contains many repetitive sequences, such as insertion elements, but none greater than 7 kbp; a PacBio can provide a complete assembly, but short reads will offer fragmented contigs Class III: Contains large repetitive sequences of >7 kb PacBio will offer a higher quality but will not be able to provide a complete genome http://genomebiology.com/2013/14/9/R101 Microbial Reference Standard • Used as a stand alone QC tool or Matrix spike • Determined DNA extract efficiency • Determine library amplification biases • Deterimine Bioinformatics parameters and cross reaction Class I Genomes ABRF MGRG Bacteria List Gram Positive Genome repeats Total genome M/O GC content Growth Methods Group BCL sim_2167 Class 1 55 5110 2564615 Staphylococcus epidermidis ATCC 12228 32.8 Standard Firma 1 sim_30 Class 1 91 5260 4170008 Halobacillus halophilus 35676 ATCC 46.8 MBA2216 Firma 1 sim_103 Class 1 65 4153 2501097 Micrococcus luteus NCTC 2665 ATCC 4698 72 Standard Actinob 1 Gram Negative Genome repeats Total genome M/O GC content Growth Methods Group BCL sim_1596 Class 1 77 5463 4639675 Escherichia coli str. K-12 substr. MG1655/atcc 700926 50.8 Standard gamma 1 sim_81 Class 1 35 5825 6845832 Pseudomonas fluorescens F113 (ATCC® 13525) 61.4 Standard Gamma 1 sim_1114 Class 1 28 5821 3850272 Pseudoalteromonas haloplanktis ATCC 14393 40.10% MBA2216 1 Pseudomonas E. coli Micrococcus Pseudoalteromonas Halobacillus Staphylococcus Oxford Nanopore Minion Data (MAP) • DNA extracted from each organism and mixed equal nMoles based on Qubit, 1 ug run in duplicate on Version 7.3 flow cells, Standard Shearing as per MAP program • Analysis of "2D" read • 534 reads for 612,038bp. Mean length was 1,146bp. Ranged from 200 to 8,299bp • MetaPhlan produced no matches. Data still too noisy. • BWA-MEM was used to force align to the six species. Ont2d Yogesh lastz • Pseudomonas flourescens: 43 • E. coli: 82 • Halobacillus halophilus: 71 • Pseudoalteromonas haloplanktis: 20 • Staphylococcus epidermidis: 3 • Micrococcus luteus: n/a DNA/RNA Extraction –Enzymes and Lasers? • Multi-lytic Enzyme Mix for Digestion of Cell Walls The MRGR is collaborating with Sigma Chemical develop a multi- enzyme product that may include • Achromopeptidase • Mutanolysin MAC4L Polyzyme • Chitinase • Lyticase • Current Extraction Reagents need higher efficiency • ANAF Filter funnels with PC NTE membranes. Metagenomic Preservatives • Have Multiple discussion with Polysciences, DNA Genotek, and NorGen Biosciences. • Testing DMSO and Ethanol currently on Lake Hillier samples. Many others. No Data Yet Extreme Microbiome Project (XMP) • What is the Extreme Microbiome Project? • A microbiome project that studies extreme and novel ecosystems • What sites are being studied • Any site that is deemed extreme and unique • Why have a dedicated Microbiome project • The MGRG efforts will need real world samples to establish standardized protocols. Extremophilic samples are perfect to worst case • What makes the project different • Whole genome shotgun sequencing PE 2x250 Illumina and Some PacBio and Oxford Nanopore. Both RNA and DNA will be included Future Sites in Antarctica include the hyper-oxide saline rich Blood Falls and Hyper-saline lakes. Expeditions of Mandy Joye (MGRG member) and Jill Mikucki (University of Tenn/Middlebury College) Blood Falls of the Taylor Valley Hyper-saline lakes of Antarctica Antarctica Results of XMP XMP Bioinformatics tools • BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) • MetaPhlAn (https://bitbucket.org/biobakery/metaphlan2) • Kraken (https://ccb.jhu.edu/software/kraken/) • PhyloSift (https://phylosift.wordpress.com/) • GOTTCHA
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