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A Viral Metagenomics Study A Viral Metagenomics Study of Honey Bee Colonyof Honey Bee ColonyCollapse DisorderCollapse Disorder
Participating Experts:W. Ian Lipkin, M.D.Professor of Epidemiology, Neurologyand PathologyMailman School of Public Health andCollege of Physicians and SurgeonsColumbia UniversityNew York, NY
Michael Egholm, Ph.D.Vice President of Research& Development454 Life SciencesBranford, CT
Sean Sanders, Ph.D.Commercial EditorScience/AAAS
Moderator:Sponsored by:
Brought to you by the Science/AAAS Business Office
Viral Metagenomics Webinar“A Viral Metagenomics Study of Honey Bee Colony Collapse
Disorder”
24 October 2007
W. Ian LipkinMailman School of Public Health and College
of Physicians and SurgeonsColumbia University
Michael Egholm454 Life Sciences, A Roche Company
An introduction to surveillance and pathogen discoveryHow and why do infectious diseases emerge and re-emerge
Agenda
Proof of causationKoch’s postulates and beyond
Staging an investigation
What can/cannot be done using with different diagnostic technologies
• Technical challenges
• Strengths and limitations
• Introduction to 454 Sequencing
A bee story…
Future perspectives
A. Fauci, NIAID/NIH
• Microbial adaptation and change
• Host susceptibility to infection(e.g. aging, HIV, cancer, transplantation, stress)
• Population growth and density
• Inadequate/deteriorating public health and regulatory infrastructure
Factors in Emerging/Re-emerging Diseases
Modified from Institute of Medicine
Factors in Emerging/Re-emerging Diseases, continued
Modified from Institute of Medicine
• Urbanization and crowding (humans & animals)
• Changing ecosystems (deforestation, global warming)
• Globalization of travel and trade
Agriculture and World Health
Global Trade in Agricultural Products
United Nations
Koch, 1890
Microbe occurs in every case of a diseaseMicrobe must be specific for that diseaseMicrobe can be isolated, grown in the laboratory and cause disease after inoculation into animals
CAUSATION: IMPLICATION OF MICROBES IN DISEASE
Potential confoundsSome microbes cannot be grown in the laboratory (Koch)Host and environmental factors may influence expression of diseaseLongterm/distant sequelae may obscure relationship to diseaseSuitable animal models may not exist (Koch)
Fredericks and Relman, 1996
Sequences should be present in most cases of a disease at sites of pathology
Few/no sequences should be present in hosts or tissues without diseaseSequences should diminish with resolution of disease and increase with relapse
Sequences should be present prior to the onset of disease
CAUSATION: IMPLICATION OF MICROBES IN DISEASE
Presence of a microbe or its genetic footprints correlate with disease (e.g. animal models, immunological responses consistent with infection, response to treatment, vaccine prophylaxis…)
A Staged Strategy for Pathogen Discovery
MassTag PCR Panels• Respiratory disease • Hemorrhagic fevers • Meningoencephalitides• Poxviruses $15/assay, 96 samples in 6 Hours
GreeneChips• Viral• Pan-pathogen • Respiratory$75/assay, 8 samples in 15 hrs
Shotgun Sequencing$5,000/assay – 1 week
QuantitativeReal-Time PCR
Consensus PCRCloning & Sequencing
Serology (IgM, increase in IgG titer)Pathology (agent distribution)Challenge experiments (reproduce disease)
454 Genome Sequencer FLX Overview
� Instant cloning by emulsion PCR� ~ 400K clonal reads � ~ 250 bp average read length� Less than 8 hours run time� MIDs allow 96 samples/plate� Average individual read accuracy >99.5% � Extremely low substitution error rate of less
than 1:10,000� Low bias from both emPCR and
Sequencing� Consensus de novo assembly >99.99% at
>10X coverage
For details and list of 90+ peer reviewed papers enabled by 454 Sequencing go to www.454.com orwww.roche-applied-science.com
454 Sequencing Overview
Shear DNA and add linkers ‘Emulsion PCR’Clonal amplification
Deposition of beads onto PTP with 1.6M wells.
Sequencing-by-synthesisDetection of PPi release
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Dramatic Cloning Bias by Conventional Sequencing Example 500 kb stretch in Listeria monocytogenes
454 coverage
Reference position (bp)
Courtesy of Drs. Nusbaum and Young of the Broad Ins titute
ABI coverage
Generation of 454 Library from DOP-PCR Product
Bio
PA BBio
Ligation
Fill inBio
Bio
Bio
1) Capture on SA-Beads & Wash2) Alkaline Elution
A B
P
Fragment end Polishing
High Throughput Sequencing
Algorithm for Reductive Analysis of Sequence DataProgram for automated identification of pathogen sequences
Outbreak of Transplant-Associated Encephalitis Identification of a novel arenavirus
Colony Collapse Disorder Profile of an emerging threat to agriculture and economic welfare
Sample preparationEnrichment for pathogen sequences
Primer
Repeat
Trimming FilteringClustering Assembly
Raw Reads
host
ContigsTrimmed Reads Unique Reads
Reductive Analysis of High Throughput Sequencing Data
Contigs and SingletsContigs and Singlets
Candidates withNucleotide Homology
Candidates withNucleotide Homology
BLASTN
Candidates withProtein Homology
Candidates withProtein Homology
BLASTX
viruses
bacteria
fungi
metazoans
parasites
Sequence � TaxonomySequence � Taxonomy
Candidate organism
Thomas BrieseSean ConlanDiana Cox-FosterLiwang CuiAndrew DrysdaleMichael EgholmDennis vanEngelsdorpJay EvansDavid GeiserEddie HolmesMady Hornig
Jeffrey HuiStephen HutchisonAbby KalksteinW. Ian LipkinVince MartinsonGustavo PalaciosJeff PettisNancy MoranPhenix-Lan QuanJan Fredrik SimonsJunhui Zhai
U of Arizona; Columbia; Penn State; USDA,FL Dept of Agriculture; PA Dept of Agriculture; and 454 Life SciencesCox-Foster et al. Science 318:283-287, 2007
Metagenomic Analysis of CCD
To Bee or Not to Bee…Colony Collapse DisorderGlobal pollination of > 90 fruit and vegetable crops$14.6B/year in US alone
Family apoidea
AlfalfaAlmondAppleApricot (some varieties)ArtichokeAvocadoBlackberryBlack locustBlueberryBoysenberryBroadbeansBroccoliBrussels sproutsBuckwheatCabbageCarawayCarrotsCatalpaCauliflowerCeleryCherryChestnutChicoryChinese cabbage
Clover (some varieties)CoconutCoffeeCollardsCorianderCottonCrabappleCranberriesCrownvetchCucumbersCurrantsDewberryDillEggplantEndiveFennelFigsFlaxGarlicGooseberriesGrapes (muscadine)GrapefruitGuarGuava
Holly KaleKenafKiwiKohlrabiLeekLespedeza (bush)Lima beansLoquatsMacadamia nutMaple (red)Mandarin orangeMangoMuskmelonsMustardNectarinesOkra OnionsOrange PapayaParsleyParsnipsPeachesPear
Pepper Persimmon (native)Plums and PrunePumpkinsRadishRapeRaspberryRutabagaSafflower SquashStrawberrySunflowerSweetcloverTangeloTangerineTendergreensTrefoilTurnipsVetchWatermelonsYucca
Colony Collapse Disorder
Collapsed colonies
Absence of adult bees, dead or alive
Presence of capped brood
Presence of food stores, not ransacked
The Clinician in Pathogen DiscoveryWest Nile virus, New York, 1999
Deborah Asnis, MD/Tracy McNamara, DVM
CCD
Diana Cox-Foster
Penn State
Normal
7 CCD operations, 6 colonies/operation, 10 bees/col ony4 Royal jelly samples
Strategy for CCD Project
RNA extractionDNAse I digestionRandom amplification
Metagenomic Sequencing/Bioinformatic Analysis
Closest sequencedrelatives identifiedby BLAST
Raw Reads
35%
33%0%
1%
0%
3%
0%
28%
Order Hymenoptera
Metazoa (Other)
Plants
Eukaryota
Fungi
Bacteria
Viruses
Unclassified
Assembled Contigs
26%
25%
3%5%
1%
8%
2%
30%
Order Hymenoptera
Metazoa (Other)
Plants
Eukaryota
Fungi
Bacteria
Viruses
Unclassified
974351668Total
27732506Unclassified
6530Viruses
2688127Bacteria
24318Fungi
99286Eukaryota
4242Plants
31991419Metazoa (Other)
3368219390
440-
Order Hymenoptera60% rRNA
ReadsContigsClassification
Raw Reads
35%
33%0%
1%
0%
3%
0%
28%
Order Hymenoptera
Metazoa (Other)
Plants
Eukaryota
Fungi
Bacteria
Viruses
Unclassified
Assembled Contigs
26%
25%
3%5%
1%
8%
2%
30%
Order Hymenoptera
Metazoa (Other)
Plants
Eukaryota
Fungi
Bacteria
Viruses
Unclassified
974351668Total
27732506Unclassified
6530Viruses
2688127Bacteria
24318Fungi
99286Eukaryota
4242Plants
31991419Metazoa (Other)
3368219390
440-
Order Hymenoptera60% rRNA
ReadsContigsClassification
35%
Raw Reads
35%
33%0%
1%
0%
3%
0%
28%
Order Hymenoptera
Metazoa (Other)
Plants
Eukaryota
Fungi
Bacteria
Viruses
Unclassified
Assembled Contigs
26%
25%
3%5%
1%
8%
2%
30%
Order Hymenoptera
Metazoa (Other)
Plants
Eukaryota
Fungi
Bacteria
Viruses
Unclassified
974351668Total
27732506Unclassified
6530Viruses
2688127Bacteria
24318Fungi
99286Eukaryota
4242Plants
31991419Metazoa (Other)
3368219390
440-
Order Hymenoptera60% rRNA
ReadsContigsClassification
33%
Raw Reads
35%
33%0%
1%
0%
3%
0%
28%
Order Hymenoptera
Metazoa (Other)
Plants
Eukaryota
Fungi
Bacteria
Viruses
Unclassified
Assembled Contigs
26%
25%
3%5%
1%
8%
2%
30%
Order Hymenoptera
Metazoa (Other)
Plants
Eukaryota
Fungi
Bacteria
Viruses
Unclassified
974351668Total
27732506Unclassified
6530Viruses
2688127Bacteria
24318Fungi
99286Eukaryota
4242Plants
31991419Metazoa (Other)
3368219390
440-
Order Hymenoptera60% rRNA
ReadsContigsClassification
28%
viruses
bacteria
fungi
metazoans
parasites
Varroa destructor mite
Kynetoplastide parasite species
Nosema sp.
Neisseria species
Gamma proteobacteria
Viruses
ssRNA (+)
Dicistroviridae
Cripavirus
Black Queen Virus
Israel acute paralysis virus
Kashmir bee virus
Iflavirus
Sacbrood virus
Deformed wing virus
Chronic bee paralysis virus
Kingdom Taxon (rank) Organism Bacteria Firmicutes (phylum) Lactobacillus sp. 1,2
Uncultured Firmicutes 2 Bacteria Actinobacteria (class) Bifidobacterium sp. 1 Bacteria Alphaproteobacteria (class) Bartonella sp. 1,2
Gluconacetobacter sp. 1,2 Bacteria Betaproteobacteria (class) Simonsiella sp. 1,2 Bacteria Gammaproteobacteria (class) Two uncultured species 1,2 Fungus Entomophthorales (order) Pandora delphacis Fungus Mucorales (order) Mucor spp. Fungus / Microsporidian
Nosematidae (family) Nosema ceranae
Fungus / Microsporidian
Nosematidae (family) Nosema apis
Eukaryota Trypanosomatidae (family) Leishmania/Leptomonas sp. Metazoan Varroidae (family) Varroa destructor Virus (unclassified) Chronic bee paralysis virus* Virus Iflavirus (genus) Sacbrood virus Virus Iflavirus (genus) Deformed wing virus* Virus Dicistroviridae (family) Black queen cell virus Virus Dicistroviridae (family) Kashmir bee virus* Virus Dicistroviridae (family) Acute bee paralysis virus Virus Dicistroviridae (family) Israeli acute paralysis virus of bees*
Bacterial Composition of CCD and Non-CCD Hives
16S rRNA analysis
Number of positive samples (% of samples tested)
Agent CCD
(n=30) Non-CCD
(n=21) Total
(n=51)
Positive Predictive
Value (%)
Sensitivity (%)
Specificity (%)
IAPV 25 (83.3) 1 (4.8) 26 (51.0) 96.1 83.3 95.2
KBV 30 (100) 16 (76.2) 46 (90.2) 65.2 100 23.8
N. apis 27 (90) 10 (47.6) 37 (72.5) 73.0 90.0 52.4
N. ceranae 30 (100) 17 (80.9) 47 (92.1) 63.8 100 19.0
All 4 agents 23 (76.7) 0 (0) 23 (45.0) 100 76.7 100
Value of CCD Candidate Markers
Real time PCR analysis of pools of up to 10 bees fr om 30 CCD and 21 non CCD operations Jan 2004 through May 2007
Work in Progress
Global distribution of IAPV
Challenge experiments in A mellifera with IAPV +/- other potential factors
Phylogenetic analyses of IAPV and KBV
Reverse genetic studies of IAPV
Characterization of other potential pathogens ident ified through metagenomics during the course of the CCD p roject
Improvements in metagenomic technology (sample preparation, throughput)
Future Application of Pathogen Discovery/Surveillance TechnologiesInfectious Causes of Chronic Disease
DiseaseCervical CA
Chronic Hepatitis/Hepatocarcinoma
Lyme Arthritis
Peptic Ulcer Disease
Whipple Disease
Bladder CA
Premature Birth
Cerebral Palsy
CauseHuman papilloma virus
Hepatitis B and C viruses
Borrelia burgdorferi
Helicobacter pylori
Tropheryma whippelii
Schistosoma haematobium
Various
Various
Compelling evidence
Future Application of Pathogen Discovery/Surveillance Technologies
Chronic Diseases Potentially Linked to Infection
DiseaseCardio/Cerebrovascular Disease
Diabetes Mellitus
Obsessive Compulsive Disorder
(PANDAS)
Schizophrenia
Inflammatory Bowel Disease
Idiopathic Pulmonary Fibrosis
Autism
Acute Macular Degeneration
CauseDental/sinus infections
Enteroviruses, other viruses
Group A streptococcus
Prenatal infections
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The future of microbes and mankind will probably unfold as episodes of a suspense thriller that could be entitled Our Wits versus Their Genes. The global scientific and public health communities must confront this reality, not only with wisdom but also with vision and sustained commitment to meet a perpetual challenge.Ó
"The future of microbes and mankind will probably unfold as episodes of a suspense thriller that could be entitled Our Wits versus Their Genes. The global scientific and public health communities must confront this reality, not only with wisdom but also with vision and sustained commitment to meet a perpetual challenge."
Josh Lederberg
A Viral Metagenomics Study of Honey Bee Colony Collapse Disorder
For more information visit: www.genome-sequencing.com or www.454.com
Q&A SessionSubmit your questions by typing them into the “Ask A Question” box at the bottom left of the console, then click t he button
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