2015ABRF Metagenomics Research Group

The Extreme Microbiome Project (XMP) and Development of New Standards for Metagenomics and Microbiome

Applications

Scott TigheUniversity 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 WildlifeDon Cater Western Australia Government

Industry Partners and Contributors

• Craig Rowell Illumina CorpSpecial Projects Coordinator

• Aaron Sin Sigma ChemicalBob Gates

• Liz Kerrigan American Type Culture CollectionJodie Lee

• Carlos Merino DNA GenotekAaron Del Duca

• Nezar Rghei Norgen Biotek Corp.

What are the Challenges with Metagenomics and where can the MGRG help ?

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 StudyEvaluating 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. Tighe121Ohio 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.

DNA Extraction Efficiency

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

Software Coverage evaluation of universal bacterial primers using themetagenomic datasets Dan-Ping Mao, Quan Zhou, Chong-YuChen and Zhe-Xue Quan BMC Microbiology 2012, 12:66

Current Challenges with Metagenomics

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 readsClass 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 contigsClass 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

Class I Genomes

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 1sim_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 1sim_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

ABRF MGRG Bacteria List

Micrococcus

StaphylococcusHalobacillus

E. coliPseudomonas

Pseudoalteromonas

• Used as a stand alone QC tool or Matrix spike• Determined DNA extract efficiency• Determine library amplification biases• Deterimine Bioinformatics parameters and cross reaction

• DNA extracted from each organism and mixed equal nMolesbased 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.

Oxford Nanopore Minion Data (MAP)

Ont2d• Pseudomonas flourescens: 43

• E. coli: 82

• Halobacillus halophilus: 71

• Pseudoalteromonas haloplanktis: 20

• Staphylococcus epidermidis: 3

• Micrococcus luteus: n/a

lastzYogesh

• Multi-lytic Enzyme Mix for Digestion of Cell WallsThe MRGR is collaborating with Sigma Chemical develop a multi- enzyme product that may include

• Achromopeptidase• Mutanolysin• Chitinase• Lyticase

• Current Extraction Reagents need higher efficiency • ANAF Filter funnels with PC NTE membranes.

DNA/RNA Extraction –Enzymes and Lasers?

MAC4L Polyzyme

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

• 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

Extreme Microbiome Project (XMP)

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)

Hyper-saline lakes of AntarcticaBlood Falls of the Taylor ValleyAntarctica

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 (https://github.com/poeli/GOTTCHA)

“Doors to Hell” Gas CraterDarvaza, Karakum Desert, Turkmenistan

Sampled by: Stefan Green (ABRF MGRG)DNA Extracted: 10 Grams yield 438 pg/ul/20ulDNASeq Library: Rubicon ThruPlex 20 cyclesSequencing: Natalia Reyero MGRG

MiSeq PE 2x250 MSU Data Analysis: Ebrahim Afshinnekoo MGRG

MetaPHan, MegaBlast

Extreme Microbiome Project

Nocardioides sp. JS614 .......................... 1 hits 1 orgs Pimelobacter simplex ............................ 1 hits 1 orgs Propionibacterium avidum 44067 .................. 1 hits 1 orgs Catenulispora acidiphila DSM 44928 .............. 1 hits 1 orgs Stackebrandtia nassauensis DSM 44728 ............ 1 hits 1 orgs Streptosporangium roseum DSM 43021 .............. 1 hits 1 orgs Leifsonia xyli .................................. 2 hits 2 orgs Streptomyces cattleya DSM 46488 ..................2 hits 1 orgs Kitasatospora setae KM-6054 ..................... 1 hits 1 orgs

The Door to Hell is noted for its natural gasfire which has been burning continuouslysince it was lit by Soviet petroleum engineersin 1971.[1] The fire is fed by the rich naturalgas deposits in the area. The pungent smellof burning sulfur pervades the area for somedistance

“Emperor Penguin Microbiome (Feces)”McMurdo Station, Antarctia

Sampled by: Vladimir Samarkin (Joye Lab)

DNA Yield: ~100 mg=36 ng/ul/30ulExtracted: MAC4L/ALO3/Omega DNASeq Library: Rubicon ThruPlex 8 cyclesSequencing: Natalia Reyero MGRG

Weill Cornell-MasonMiSeq PE 2x250 MSU

Data Analysis: Ebrahim Afshinnekoo MGRG

Extreme Microbiome Project

• Isolated from deep-sea sediment of the Mariana Trench • Grow in aquatic environments• Petroleum reservoirs• Cool soil environments• Withstand pH of 2-12, temperature of 5-78 degrees Celsius, and 0 to 30% salinity

Geobacillus kaustophilus

Clostridium perfringens• Normal component of decaying vegetation, marine sediment, the intestinal tract of

humans and other vertebrates, insects, and soil.

• Third most common cause of food poisoning in the United Kingdom and the United States though it can sometimes be ingested and cause no harm.

• α-toxin producing strains are ausative agent of Gangrene• Anaerobic bacteria

Lake Hillier Australia’s Recherche Archipelago

20ul Sediment 20ul WaterMarine Agar 2216-2% NaCl [oops]

• Extreme Hyper saline shallow lake- 25% during sampling• Salt precipitates out of solution instantly• pH 7.4 at 26C

Lake Hillier Australia’s Recherche Archipelago

Tested three collection Preservatives ETOH, DMSO. Fresh (cold)

Extracted RNA (Trizol LS) DNA (MAC4L-Omega) Tested two processing protocols

Diluted and FilteredDiluted and Centrifuged

Method Sample Volume (mL)Total RNA

(25ul)Total DNA

(25ul)Sed-Fresh-filtered 0.5 ND 7.75Sed-ETOH-filtered 1.7 50.75 192.5Sed-DMSO-filtered 1.7 35 327.5Water-Mid-fresh-filtered 7.5 27.5 23.3Water-Mid-ETOH-filtered 7.5 ND 10.0Water-Mid-DMSO-filtered 7.5 ND 105.0Sed-Fresh-Direct 0.2 55 55.0Sed-ETOH-Direct 0.2 37.5 15.0Sed-DMSO-Direct 0.2 37.5 97.5Bank-Fresh-Direct 0.2 NA 627.5Bank-ETOH-Direct 0.2 950 520.0Bank-DMSO-Direct 0.3 NA 560.0

Filter Process

Direct Fresh DMSO ETOH

Summary

Shotgun metagenomics is a great tool for identification purposes but not for microbial shift studies, when 16s is more applicable.

Microbial standards are absolutely necessary and will be available soon

Multi-enzyme digestion reagents will be available soon

The extreme microbiome is a great study tool for optimizing metagenomics methods as worse case scenario.

Thank you for your Attention

Would you like to join the MGRG?Please visit our website:

www.extrememicrobiome.org

Please visit our Poster# 266 Monday 100pm