Use of Next Generation Sequencing

(Metagenomics) in Type 1 Diabetes

Joseph Petrosino, Ph.D.
International Society for Pediatric and
Adolescent Diabetes
September 5th, 2014

No Relevant Conflict of Interest

Funding sources:

National Institute of Diabetes and Digestive and Kidney Diseases

(NIDDK)
National Institute of Allergy and Infectious Diseases (NIAID)
National Institute of Environmental Health Sciences (NIEHS)
National Human Genome Research Institute (NHGRI)
Juvenile Diabetes Research Foundation (JDRF)
Cancer Prevention Research Institute of Texas (CPRIT)
Alkek Foundation
Houston Men of Distinction (Benefiting childhood diseases)

The Texas Medical Center:

A Microbiome Goldmine

Member Institutions: 52
26 agencies of government
26 private not-for-profit health-related institutions
Annual Patient Visits: 7.1 million
Annual International Patient Visits: 16,000
Employees: 92,500
Full-time Students: 34,000*
Volunteers Daily: 12,000
Residents and Fellows: 4,000

Visiting Scientists, Researchers and Students: 7,000**

Total Hospital Beds: 6,900
Total Bassinets: 400
Annual Surgeries: 350,000
Babies Delivered (Annually): 28,000
Total Budget (All Institutions): $14 billion
Total Size/Gross Square Foot (All Campuses):
45.5 million sf
1,300 Acres
280 Buildings

The Texas Medical Center:

A Microbiome Goldmine

MILLIONS OF SAMPLES
Member Institutions: 52
26 agencies of government
26 private not-for-profit health-related institutions
Annual Patient Visits: 7.1 million
Annual International Patient Visits: 16,000
Employees: 92,500
Full-time Students: 34,000*
Volunteers Daily: 12,000
Residents and Fellows: 4,000

Visiting Scientists, Researchers and Students: 7,000**

Total Hospital Beds: 6,900
Total Bassinets: 400
Annual Surgeries: 350,000
Babies Delivered (Annually): 28,000
Total Budget (All Institutions): $14 billion
Total Size/Gross Square Foot (All Campuses):
45.5 million sq ft
1,300 Acres
280 Buildings

The metagenomic approach

Sample Preparation

Sample

Sequencing and Analysis

16S rRNA gene

sequencing

WGS on DNA

Enrich bacteria,
viruses, eukes

WGS on cDNA

Community structure

Bacterial pangenome
DNA viruses

Bacterial transcriptome

Extract
DNA/RNA
WGS on cDNA
from virus prep

Virome and more

Alkek Center for Metagenomics and

Microbiome Research
Mission: To understand how the microbiome impacts health and disease, to translate
this understanding for better therapeutics and diagnostics, and to serve as a hub for
these activities internationally
Enrich established studies and develop new projects

Advance sequencing, culturing, and analysis technologies

Enable and fund feasibility/pilot studies
Develop ties with clinicians to enhance translational impact

Develop means to study/test host-microbiome interactions

Advance animal and microbial model systems

Murine, primate-hypothesis-driven questions

Germ free facility experiments

Host genome wide association studies (GWAS) combined with microbiome analysis

Develop/recruit critical mass for systems biology approaches to microbiome

research

Genetics, immunology, biochemistry, cell biology, metabolomics

Translate findings to the clinic

Collaborators: Human samples

Ocular Surface
BCM
National Eye Institute

Skin
BCM
MD Anderson
Texas Childrens

Blood
BCM
MD Anderson

Oral
UT Health Science Center
UT School of Dentistry
Multi-site
U. Texas Health Science Center
U. Florida
Miami U
U. Texas School of Public Health
Sam Houston State U
U. Colorado
U. South Florida

Reproduction/Urogenital
BCM
Michigan State
North Shore University Hospital
Stony Brook University
Texas Childrens
University of South Carolina

Lung/Airways
BCM
Gastrointestinal
Harvard Med. School
BCM
Mass. General Hospital
Texas Childrens
U. Michigan
Harvard Med. School
UT Health Science
National Institute of Health Center
Texas A&M HSC
Panola College
U. Javeriana
City of Hope
Lexicon Pharmaceuticals
Michigan State
Nazarbayev U. (Kazakhstan)
U. del Norte (Colombia)
U. Louisville
Peruvian University Cayetano Heredia
Sam Houston State University
Nottingham U. (UK)
U. Texas School of Public Health
UT Medical Branch
MD Anderson

Collaborators: Animal Models

Ocular Surface
BCM
Gastrointestinal
BCM
U. Michigan
Iowa State U.
U.C. Irvine
Harvard Med. School
Texas A&M HSC
Methodist Research Inst.
U. Texas Medical Branch
Lexicon Genetics
Albany Medical College
U. Utah
MD Anderson
UH College of Pharm.
NIH
Sam Houston State U.

Disease and Disease Model Projects

> 200 projects and growing

Chronic Kidney Disease (Raj, George Washington University)

NASA extreme environments (JPL, Pasadena)
Autism (Mazmanian, Cal Tech)
Uveitis (Caspi, NIH/NEI)
Type 1 Diabetes (Krischer; Burkhardt, USF; Atkinson, UF, Dominguez-Bello, NYU)
Microbiome of death (Bucheli and Lynne, SHSU)
Microbial surveillance (Klotman (BCM-MC); Maresso (school), BCM)
Synthetic probiotic organisms (Tabor, Rice)
Necrotizing enterocolitis (Burrin; Premkumar, BCM)
Fecal transplants (Graham, BCM; Wilkerson, MD Anderson; ASMCUE, SHSU)
HIV (Vigil, UT; Bryson, UTH; San Juan, U. Norte; Klotman, BCM)
Colon cancer (El Serag, BCM; Daniel-MacDougall, MD Anderson)
Type 2 Diabetes (Fisher-Hoch, UTSPH)
COPD cancer (Liu, BCM)
Clostridium difficile (Graham and Koo, BCM; DuPont, UTSPH)
IBS/IBD (DuPont, BCM (adults); Versalovic, TCH/BCM (children); Dann UTMB; Round, Utah)
Leukemia (Adachi, MD Anderson)
Pancreatic cancer and cachexia (Fogelman, MD, MDACC)
Cannabis abuse, withdrawal, and recovery (Verrico, BCM-VA)
Anorexia and bulimia (Pinho, Albert Einstein Medicina Diagnstica, Brazil)
Suicide and suicidal ideation (Salas, BCM)

Proposed progression mechanism for T1D

(Precipitating Event?)

Beta cell mass

Genetic
Predisposition

Overt
immunologic
abnormalities

Normal insulin
release

Progressive
loss insulin
release
Glucose
normal

Overt
diabetes

C-peptide
present
No C-peptide

Age (years)

Eisenbarth et al. NEJM 1986

Known and hypothesized

contributing factors toward T1D
Risk Factors
HLA
Relative with T1D
Race/Ethnicity
Geography
Environmental Exposures
Viruses (Enterovirus)
Infectious Agents
Diet (breast feeding, cows
milk, gluten, vitamin D)
Seasonality
Perinatal factors (mode of
delivery, social environment)
Obesity
Stress

Islet Autoimmunity

T1D

Known and hypothesized

contributing factors toward T1D
Risk Factors
HLA
Relative with T1D
Race/Ethnicity
Geography
Environmental Exposures
Viruses (Enterovirus)
Infectious Agents
Diet (breast feeding, cows
milk, gluten, vitamin D)
Seasonality
Perinatal factors (mode of
delivery, social environment)
Obesity
Stress

Islet Autoimmunity

T1D

Known and hypothesized

contributing factors toward T1D
Risk Factors
HLA
Relative with T1D
Race/Ethnicity
Geography
Environmental Exposures
Viruses (Enterovirus)
Infectious Agents
Diet (breast feeding, cows
milk, gluten, vitamin D)
Seasonality
Perinatal factors (mode of
delivery, social environment)
Obesity
Stress

Islet Autoimmunity

x
T1D

Clinical Centers

Principal Investigators
ke Lernmark, Ph.D.Lund U.
Jeffrey Krischer, Ph.D.U. South Florida
William Hagopian, M.D., Ph.D. U. Washington
Olli Simell, M.D., Ph.D. U. of Turku
Jorma Toppari, M.D., Ph.D.-- U. of Turku
Anette Ziegler, Ph.D. --Technische Universitt
Mnchen
Marian Rewers, M.D., Ph.D. U. Col. Denver
Jin-Xiong She, Ph.D.Jinfiniti Biosciences
Beena Akolkar, Ph.D.NIH/NIDDK

NIDDK
NIAID
NICHD
NIEHS
CDC
JDRF

Overarching TEDDY project goals

Identify environmental factors and gene-environment
interactions causing islet autoimmunity and T1D
Explore differences in the environmental determinants
of T1D across diverse populations and ethnic groups in
children with and without first-degree T1D relatives

Study the immunopathogenesis and the natural history

of T1D from birth
Establish central repository of data and biological
samples for additional hypothesis based research

TEDDY: The Environmental Determinants of

Diabetes in the Young
Coordinating Center PI: Jeff Krischer, University of South Florida

Project description:

Recruit >8,500 neonates across six clinical centers in US and Europe

Newborns from the general population with genetic risk (~90% of cohort) and those with first-degree
relatives of probands with T1DM (~10% of cohort)

Children followed for up to 15 years for the appearance of various beta-cell

autoantibodies and diabetes

Documentation of early childhood diet, reported and measured infections,

vaccinations, antibiotics, psychosocial stressors, and MUCH more

Stool collected monthly through age 4, then quarterly through age 10, then
biannually through age 15

Blood collected every quarter

Comprehensive microbiome study for TEDDY

Largest clinical microbiome study to date: ~40Tb of raw data

provides unique opportunities and challenges

Primary goals: what are we doing
Identify viral candidates that may trigger T1D (stool, plasma, PBMC)
- Viral metagenomics including virus-targeted extraction arm
- Culture-based viral amplification arm to enrich viruses
Associate microbiome (bacterial, eukes, virus) changes with progression
to T1D
- 16S, 18S, WGS, RNA viral (stool and plasma)
- Identify metagenomic biomarkers associated with T1D

Contract to CMMR and R. Lloyd (BCM-MVM) for data generation and

analysis coordination (9/2012-8/2015)

Comprehensive microbiome study for TEDDY

Largest clinical microbiome study to date: ~40Tb of raw data

provides unique opportunities and challenges

Primary goals: what are we doing
andtrigger
triggers
will plasma,
lead to:
Microbial
Identify viralassociations
candidates that may
T1D (stool,
PBMC)
Viral metagenomics
including virus-targeted extraction arm
- more
sensitive diagnostics
Culture-based
viral amplification arm to enrich viruses
- new
therapeutics
Associate microbiome (bacterial, eukes, virus) changes with progression
to T1D
earlier interventions
- possibly
a cure
(eg.
vaccine
against the trigger)
16S, 18S, WGS,
RNA
virala(stool
and plasma)
- Identify metagenomic biomarkers associated with T1D

Contract to CMMR and R. Lloyd (BCM-MVM) for data generation and

analysis coordination (9/2012-8/2015)

Nested Case-Control Design for TEDDY Microbiome Lab

419 cases of persistent confirmed islet antibodies

114 cases of T1D

1:1 matching cases : controls

Longitudinal stool samples (n=13,403)
Longitudinal plasma samples (n=6,380)

Nasal swab samples also available

Update 9/2014 (about 2/3 complete)

Progress as of 9/2014

Stool

19,783 Total Samples

Cultured
Total Extracted 16S 18S/ITS WGS Virome Virome
12,67
13,403 13,403*
2
0
11,882 8,589
4,055

Plasma
1,469
* Some
samples on6,380
re-order for re-extraction.

N/A

N/A

1,469 1,469

316

Highlights
All stool samples have had nucleic acids extracted using
novel robotic pipelines with >95% success rate
Extraction and sequencing controls with every run
Over 6.8 Tb of WGS data thus far (HMP 3.6 Tb)
> 10,000 reads per 16S rDNA sample
> 1.1 Tb of direct virome data
Attempting to culture viruses from every sample
Microbial eukaryote (18S) arm about to begin
Ajami, Ayvaz, Bauch, Kusic, Railey, Tamegnon

The TEDDY 50
In 3/2014 TEDDY leadership requested an analysis teaser to present to the
consortiumthey provided limited metadata for 50 subjects
and 4 days

What does the data look like so far

Is the data different than would be expected given the age of the TEDDY
children studied and other populations that have been studied

What is the best way to think about using the data in conjunction with other
TEDDY data on genetics and environmental exposures

MetabolomicsFiehn Laboratory, UC Davis Genome Center

ProteomicsPacific Northwest National Laboratory
Human gene expression (plasma) Jinfiniti Biosciences, LLC

The following are highlights from these analyses

Wong, Smith, and Ajami

TEDDY 50 metadata provided

Metadata
Gender
FDR
Age in months
HLA Category
Country
Collection date
NOT clinical endpoints

TEDDY 50
50 subjects
567 samples

HLA genotypes
1='DR4*030X/0302*DR3*0501/0201 (very high risk; 1/15 get T1D by 15 years)**
2='DR4*030X/0302*DR4*030X/0302 (high risk; 1/20-1/30 get T1D by 15 years)**
4='DR4*030X/0302*DR8*0401/0402 (high risk, more genotypes from Finland)**
5='DR4*030X/0302*DR1*0101/0501 (FDR high risk, Gen. pop. with this genotype not enrolled)
6='DR4*030X/0302*DR13*0102/0604 (FDR high risk, Gen. pop. with this genotype not enrolled)
9='DR3*0501/0201*DR3*0501/0201 (moderate risk; maybe 1/35-1/50 get T1D by 15 years;
also highest risk for Celiac disease)

**All enrolled in TEDDY regardless of First Degree Relative (FDR) status

Bacterial Associations
16S rRNA gene
and
WGS data

Alpha Diversity (Bacterial community diversity)

16S:QIIME;UPARSE;Full SILVA DB
Age, HLA type, and
country had a
significant correlation
with observed alpha
diversity within
samples.

Country: Pairwise T-Test

HLA: Pairwise T-Test

1

2 0.03400

Germany 0.00068

4 4.8E-08

1.1E-10

Sweden 0.00845

0.09162

US 0.01568

0.01657

0.22634

0.00024

0.00309

9 0.43198

Finland Germany

Gender

First Degree Relative

HLA Category

Subject ID

Age in Years

Sampling Date

Sweden

Country

Birth month

Microbial community shifts from birth

Weighted UniFrac Analysis by Age
Age (months)
2

32

59

PCoA

With the full data set, TEDDY may provide the best view of the maturation of the infant GI microbiome,
with data from multiple countries
Weighted UniFrac analysis suggests multiple starting community structures projecting towards a single structure

WGS data highlight nature of shifts in developing microbiome

25 most abundant taxa

Do these patterns hold true in all children, or only those progressing to T1D?

Keystone organisms associated with age

WGS data reveals HLA specific microbiome signatures

HLA genotypes
1='DR4*030X/0302*DR3*0501/0201'
2='DR4*030X/0302*DR4*030X/0302
4='DR4*030X/0302*DR8*0401/0402
5='DR4*030X/0302*DR1*0101/0501
6='DR4*030X/0302*DR13*0102/0604
9='DR3*0501/0201*DR3*0501/0201

25 most abundant taxa

WGS: Metabolic pathways

Subject

Preliminary viral data in TEDDY

Obtained from the bacterial WGS data

Additional data tomorrow morning: Dr. Heikki Hyty

Evidence of Enteroviruses in T1D

RNA

anti-VP1

T1D

Healthy

Oikarinen, et al. Clin Exp Immunol. 2008 January; 151: 7175

WGS: DNA eukaryotic viruses

Bocavirus
HBoV SH3
HBoV 2

Herpesvirus 4
Herpesvirus 5

Alphapapillomavirus

Shallow WGS Sequencing of TEDDY stool samples (1Gb)

Adenovirus
AdV3
AdV6
AdV31
AdV41
AdV61
AdV Group A
AdV Group B
AdV Group C
AdV Group F

Virus hunting in longitudinal samples

One subject
8 samples
877,904 reads mapped to 5 viral genomes
Ref. genome

Ref. genome
Size

Mapped
reads#

Genome
Coverage

Human adenovirus 41 isolate NIVD103

34169

396568

97%

Vaccinia virus GLV-1h68

203057

108

1.7%

Human adenovirus 41 isolate Tak

34188

462778

99.6%

Human adenovirus F (L19443.1)

34214

9402

13%

Human adenovirus F (NC_001454.1)

34214

9048

14%

Extracting whole virus genomes from WGS data

Reads #

Pooling of longitudinal samples for one subject

462778 reads assembled into 1 contig of 34,033 bp.
Contig matches Human adenovirus 41 isolate Tak with an
average of ~1354x coverage.
Contig covers 99.5% of the ref. genome (34,188 bp).

Genome position (bp)

Tian

Another approach to virus

hunting in T1D
Network of Pancreatic Organ Donors (nPOD)-Virus Study

CMMR Viral Metagenomic Pipeline

Sample
Log in and
storage (-80C)

Ambion MagMax Viral

RNA/DNA (LifeTechnologies)

Automated Nucleic Acid

extraction and QC

Random
amplification,
barcoding, and
pooling

Illumina
Sequencing

Analysis
Pipeline

10mer semi-random primer with an integrated 12mer barcode.

94C 2min Initial denaturation
94C 30sec denaturation
33C 30sec (10% RS) annealing
58C 30sec extension
68C 5min Final extension

50X

Illumina HiSeq 2500 (2x150)

3 samples/lane

CMMR Pipeline for Viral Metagenomics (cont.)

Sequencing
Illumina HiSeq 2500
3 samples/lane

Trimming, custom
de-multiplexing ,and
low complexity
filtering

HG20, NCBI Bacterial RefSeq,

and UniVec + EMVEC

CMMR custom viral nucleotide database,

86% seq.id (NCBI RefSeq, EBI, and PHAST)

Custom CMMR pipeline*

Read Coverage
Read span
Read pair concordance

Remove host,
human, bacterial,
and vector
sequences

Removal 10mer semi-random primer

with an integrated 12mer barcode.
Remove Illumina adapter, DUST and entropy
filtering, and barcode and primer removal

Translated
nucleotide query
(USEARCH)

CMMR custom viral protein database (>80%

seq. id)

Nucleotide query
(Bowtie2)

Determine quality
and coverage of
positive hits

Constraints
1 mapped paired-end read per sample
50 bases aligned to reference genome
Both ends of the pair must hit the same genome(s)

DNA/RNA Viruses and Phage are Captured

Eukaryotic viruses
-Found in
all subjects
Patterns emerging
Assembly helps
need to verify hits
colonizing?
intact?

Body Site
Comparisons

Phage

Sensitivity limited by depth of sequencing

(and tools for analysis)

3 Gb

300 Mb

30 Mb

Sequencing Depth--Illumina
Mock community of Enteroviruses in PBMC

Metagenomic vs qPCR Sensitivity

With Rick Lloyd (BCM) and Heikki Hyty (U. Tempere)

QCMD (Quality Control for Molecular Diagnostics)

Test Sample Content for Blinded QC

http://www.qcmd.org/

QCMD Barcodes and Data Yield

SAMPLE
QCMD-1
QCMD-2
QCMD-3
QCMD-4
QCMD-5
QCMD-6
QCMD-7
QCMD-8
QCMD-9
QCMD-10
QCMD-11
QCMD-12

BARCODE
TCCCTTGTCTCC
ACGAGACTGATT
ATCACCAGGTGT
ATCGCACAGTAA
AGCGGAGGTTAG
TACAGCGCATAC
ACCGGTATGTAC
AATTGTGTCGGA
AGTCGAACGAGG
ACCAGTGACTCA
CAGCTCATCAGC
GCAACACCATCC

** TEDDY low coverage target is 1Gb/sample

Sample ID

Yield (Gbases)

QCMD1

0.25

QCMD2

1.22

QCMD3

1.43

QCMD4

4.0

QCMD5

4.7

QCMD6

3.46

QCMD7

0.51

QCMD8

0.06

QCMD9

3.83

QCMD10

4.45

QCMD11
QCMD12

1.19
2.1

QCMD Sequencing Results

Sample ID

Data retrieved (Gb)

Result

QCMD1

0.25

Human Enterovirus B3

QCMD2

1.22

Echovirus 30

QCMD3

1.43

Coxsackie A9

QCMD4

4.0

Echovirus 11

QCMD5

4.7

Echovirus E11

QCMD6

3.46

Enterovirus 68

QCMD7

0.51

Enterovirus 83

QCMD8

0.06

No enteroviral hits

QCMD9 (NEG CTL)

3.83

No enteroviral hits

QCMD10
QCMD11

4.45
1.19

Coxsackievirus B3
No enteroviral hits

QCMD12

2.1

Coxsackievirus A24

Sensitivity directly related to sequencing depth and virus abundance (see next slide)

QCMD Sequencing Results

107

107

4/636 Primers
qPCR
positive only

106

105
Viral copies/ml

Viral copies/ml

106

104
103
102
101
100

Seq. and qPCR

positive

105
104
103

Bar-graph shows qPCR and

seq
results
4/636 Primers
Below shows sequencing of
some of those products (top
row) and metagenomic results
(bottom row)

102
101

13-01 13-02 13-03 13-04 13-05 13-06 13-07 13-08 13-09 13-10 13-11 13-12

100

Sanger
Sequencing of
qPCR product
(R. Lloyd)

Illumina
Sequencing
(70% or 95%
seq.id)

13-01 13-02 13-03 13-04

13-05 13-06 13-0713-06
13-08 13-09 13-10 13-11
13-12
13-04
13-05
13-07

13-01

13-02

13-03

Not
sequenced

Echovirus 30

Not
sequenced

Not
sequenced

Enterovirus B87,
CxB1, CxB4, and
Echovirus 30

Not
sequenced

Human
Enterovirus
B
3 paired
reads

Echovirus 30
(95% seq. id)
64 Paired
reads

Echovirus 24,
and CxA9
(80% seq. id)

Echovirus 11,
Enterovirus
79, and CxA9
(95% seq. id)

Echovirus E11
1 paired reads

Human
Enterovirus 68
(95% seq. id)
403 pairedreads

13-08

13-09

13-10

13-11

13-12

Not
sequenced

Not
sequenced

No hit

Enterovirus 71
and CxA6

CxB4 and
Enterovirus B

Not
sequenced

Echovirus 3 (80%
seq. id)

No hit

No hit

CxB3*

No hit

Enterovirus
C104 (70%
seq. id)

Sequencing reads were mapped against CMMR custom viral database at high and low stringency levels,
80%-95% sequence identity.
Constraints
1 mapped paired-end read per sample
50 bases aligned to reference genome
Both ends of the pair must hit the same genome(s)

nPOD-V Mission
nPOD is a JDRF sponsored repository for pristine human
tissues collected from T1D individuals post-mortum
Identify viral nucleic acids associated with nPOD tissues
using NexGen Sequencing and qPCR based approaches
Improve the technologies that will make this possible
Can we find a candidate viral trigger(s)?
Mark Atkinson (UF)--nPOD
Alberto Pugliese (U Miami)nPOD-V
And help of many others!

BCM/CMMR Pipeline for Viral Metagenomics

Sample

Human DNA/RNA depletion

Nucleic Acid
extraction

And/Or
Virus enrichment

Random
amplification,
barcoding, and
pooling

Illumina
Sequencing

Analysis
Pipeline

Ambion MagMax Viral RNA/DNA

(LifeTechnologies)

semi-random primer
with an integrated
barcode

Illumina HiSeq 2500

(2x100)
3 samples/lane

Depletion studyfirst trial

Following depletion optimization strategies, attempt to isolate viruses from nPOD
pancreatic tissue from 10 donors.compare with and w/o depletion approach

Human depletion strategy resulted in a ~6% drop in human reads

on average (16% max)
More human depleted samples contain hits to viruses.
9/10 subjects had Human Herpesvirus 6B hits. 0/10 in controls

Other viruses found include Human Parvovirus B19, Torque teno

virus, Adenovirus C, Human papillomavirus 49, and Human
Herpesvirus 7

Revealed most in depleted sample pairs

Compared to controls

Human Herpesvirus 6B hits

All samples
Human Herpesvirus 6B complete genome (162kb)
AB021506

Human Herpesvirus 6B is found in nearly 100% of adults and is

associated with systemic manifestations of pancreatitis, hepatitis, and
intestinal or meningeal encephalitis (Descamps et al. 2003).

Summary and future directions

Unprecedented clinical microbiome project associated with TEDDY cohort is
well underway
Already surpasses Human Microbiome Project dataset in size
To be completed in Fall 2015
Microbiome analysis to be integrated with metabolomics, proteomics, host gene
expression

Even pilot analyses are providing unique insights in the maturation of the
microbiome from birth in infants from US and Europe
Associations with HLA are provocative and may underlie autoimmunity states

Deepest hunt for viral triggers for T1D is just beginning in two projects
Only DNA data for TEDDY thus far, RNA data pending (e.g. Enteroviruses)
Provocative hits in nPOD project

Further optimization of virus enrichment (capture/depletion) strategies will

further aid in the hunt for T1D triggers
Laser capture microscopy will be used to select tissues to sequence in future rounds of
nPOD analysis

Acknowledgements
Nadim Ajami
Russ Carmical
Elicia Grace
BCM MVM
Xiangjun Tian
Richard Lloyd
Daniel Smith
Michael Holder
Lauren Railey
Lenka Kusic
Matthew Ross
Tulin Ayvaz
Tonya Bauch
Lisa Atkins
Matthew Wong
Auriole Tamegnon
Tatiana Fofanova
Tyler McCue
Megan Coombs
Lorenzo DAmico
Diane Smith Hutchinson
Nguyen Truong

Richard Gibbs
Donna Muzny
Ginger Metcalf
Harsha Doddapaneni

TEDDY and nPOD

ke Lernmark (Lund)
Jeffrey Krischer (USF)
William Hagopian (UW)
Olli Simell (Turku)
Jorma Toppari (Turku)
Anette Ziegler (TUM)
Marian Rewers (UCol)
Jin-Xiong She (Jinfiniti)
Beena Akolkar (NIDDK)
Mark Atkinson (UF)
Alberto Pugliese (UMiami)
Heikki Hyty (Tempere)