Tracking Immune Biomarkers and the Human Gut Microbiome: Inflammation, Crohn's Disease, and Colon Cancer
“Tracking Immune Biomarkers andthe Human Gut Microbiome:Inflammation, Crohns Disease, and Colon Cancer”USC Monthly Seminar SeriesPhysical Sciences in Oncology CenterLos Angeles, CAMay 17, 2013Dr. Larry SmarrDirector, California Institute for Telecommunications and InformationTechnologyHarry E. Gruber Professor,Dept. of Computer Science and EngineeringJacobs School of Engineering, UCSD1
AbstractColon Cancer is the most common cancer among Inflammatory Bowel Disease (IBD)patients and IBD is one of the three leading high-risk factors for Colon Cancer. In 2012 itwas found, by using genetic sequencing of the gut microbiome, that Fusobacteriasequences were enriched in colorectal carcinomas (CRC). To explore this possible linkbetween inflammation, gut microbes, and colon cancer I have turned my own body into a"genomic observatory." I have been tracking over 100 blood/stool biomarkers in my ownbody every few months for the last five years, with a focus on immune variables. Usingkey biomarkers and imaging technologies I diagnosed myself as having late-onsetCrohns Disease, one of the two forms of IBD. Besides obtaining one million SNPs of myhuman genome, I have collaborated with the J. Craig Venter Institute to metagenomicallysequence my gut microbiome at three different times during a period of highinflammation. My microbiome was compared with 50 other subjects, sequenced by theNIH Human Microbiome Project--35 healthy and the remainer with IBD. I discovered thatat the height of my inflammation (CRP~30), I had 8% relative abundance of Fusobacteria,40x healthy subjects. Following antibiotic/corticosteroid therapy the Fusobacteria werereduced 90-fold. The next step is to move to high-throughput integrated personal"omics" to refine the host-microbiome dynamics. With these new tools ofcomputationally-intensive omics, there is a hope that we will gain new insights into thepathogenisis of CRC.
Visualizing Time Series of150 LS Blood and Stool Variables, Each Over 5-10 YearsCalit2 64 megapixel VROOM
Only One of My Blood MeasurementsWas Far Out of Range--Indicating Chronic InflammationNormal Range<1 mg/LNormal27x Upper LimitAntibioticsAntibioticsEpisodic Peaks in InflammationFollowed by Spontaneous DropsComplex Reactive Protein (CRP) is a Blood Biomarkerfor Detecting Presence of Inflammation
Lactoferrin is an Antibacteria GlycoproteinShed from WBC Neutrophils Into Stool SampleNormal Range<7.3 µg/mL124x HealthyUpper LimitAntibioticsAntibioticsLactoferrin Sequesters IronTypicalLactoferrinValue forActiveIBD
Colonoscopy Images ShowInflamed Pseudopolyps in 6 inches of Sigmoid ColonDec 2010 May 2011
Descending ColonSigmoid ColonThreading Iliac ArteriesMajor KinkConfirming the IBD (Crohn’s) Hypothesis:Finding the “Smoking Gun” with MRI ImagingI Obtained the MRI SlicesFrom UCSD Medical Servicesand Converted to Interactive 3DWorking WithCalit2 Staff & DeskVOX SoftwareTransverse ColonLiverSmall IntestineDiseased Sigmoid ColonCross SectionMRI Jan 2012
MRE Reveals Inflammation in 6 Inches of Sigmoid ColonThickness 15cm – 5x Normal Thickness“Long segment wall thickeningin the proximal and mid portions of the sigmoid colon,extending over a segment of approximately 16 cm,with suggestion of intramural sinus tracts.Edema in the sigmoid mesenteryand engorgement of the regional vasa recta.”– MRI reportClinical MRISlice ProgramDeskVOX 3D ImageCrohns diseaseaffects the thicknessof the intestinal wall.Having Crohns diseasethat affects your colonincreases your riskof colon cancer.
An MRI Shows Sigmoid Colon Wall ThickenedIndicating Probable Diagnosis of Crohn’s Disease
Why Did I Have an Autoimmune Disease like IBD?Despite decades of research,the etiology of Crohns diseaseremains unknown.Its pathogenesis may involvea complex interplay betweenhost genetics,immune dysfunction,and microbial or environmental factors.--The Role of Microbes in Crohns DiseasePaul B. Eckburg & David A. RelmanClin Infect Dis. 44:256-262 (2007) So I Set Out to Quantify All Three!
I Wondered if Crohn’s is an Autoimmune Disease,Did I Have a Personal Genomic Polymorphism?From www.23andme.comSNPs Associated with CDPolymorphism inInterleukin-23 Receptor Gene— 80% Higher Riskof Pro-inflammatoryImmune ResponseNOD2ATG16L1IRGMNow Comparing163 Known IBD SNPswith 23andme SNP Chip
Fine Time Resolution Sampling Reveals DistinctDynamics of Innate and Adaptive Immune SystemNormalNormal
Four Immune Biomarkers Over TimeCompared with Four Signs/SymptomsHere Immune biomarkers are normalized 0 to 1,with 1 being the highest value in five yearsSource: Photo of Calit2 64-megapixel VROOM
To Map My Gut Microbes, I Sent a Stool Sample tothe Venter Institute for Metagenomic SequencingGel Image of Extract from Smarr Sample-Next is Library ConstructionManny Torralba, Project Lead - Human Genomic MedicineJ Craig Venter InstituteJanuary 25, 2012Shipped Stool SampleDecember 28, 2011I Receiveda Disk Drive April 3, 2012With 35 GB FASTQ FilesWeizhong Li, UCSDNGS Pipeline:230M ReadsOnly 0.2% HumanRequired 1/2 cpu-yrPer Person Analyzed!SequencingFundingProvided byUCSD School ofHealth Sciences
CAMERA and NIH Funded Weizhong Li Group’s MetagenomicComputational NextGen Sequencing PipelineRaw readsRaw readsReads QCHQ reads:HQ reads:Filter humanBowtie/BWA againstHuman genome andmRNAsBowtie/BWA againstHuman genome andmRNAsUnique readsUnique readsCD-HIT-DupFor single or PE readsCD-HIT-DupFor single or PE readsFurther filteredreadsFurther filteredreadsFiltered readsFiltered readsFilter duplicateCluster-basedDenoisingCluster-basedDenoisingContigsContigsAssembleVelvet,SOAPdenovo,Abyss-------K-mer settingVelvet,SOAPdenovo,Abyss-------K-mer settingContigs withAbundanceContigs withAbundanceMappingBWA BowtieBWA BowtieTaxonomy binningTaxonomy binningFilter errorsRead recruitmentFR-HIT againstNon-redundantmicrobial genomesFR-HIT againstNon-redundantmicrobial genomesVisualizationVisualizationFRVtRNAsrRNAstRNAsrRNAstRNA-scanrRNA - HMMORFsORFsORF-finderMegageneNon redundantORFsNon redundantORFsCore ORF clustersCore ORF clustersCd-hit at 95%Cd-hit at 60%Protein familiesProtein familiesCd-hit at 30% 1e-6FunctionPathwayAnnotationFunctionPathwayAnnotationPfamTigrfamCOGKOGPRKKEGGeggNOGPfamTigrfamCOGKOGPRKKEGGeggNOGHmmerRPS-blastblastPI: (Weizhong Li, UCSD):NIH R01HG005978 (2010-2013, $1.1M)
Additional Phenotypes Added from NIH HMPFor Comparative Analysis5 Ileal Crohn’s, 3 Points in Time6 Ulcerative Colitis, 1 Point in Time35 “Healthy” Individuals1 Point in TimeDownload Raw Reads~100M Per Person
We Computationally Align 230M Illumina Short ReadsWith a Reference Genome Set & Then Visually Analyze~4500 Reference Genomeswith Strains and Viruses
From Taxonomy to Function:Analysis of LS Clusters of Orthologous Groups (COGs)Analysis: Weizhong Li & Sitao Wu, UCSD
Gut Microbiome Metagenomic Analysis:From Short Reads to Taxonomic and Gene Diversity• Analyzed Healthy and IBD Patients:– LS, 13 Crohns Disease &11 Ulcerative Colitis Patients,+ 150 HMP Healthy Subjects• Gordon Compute Time– ~1/2 CPU-Year Per Sample– > 200,000 CPU-Hours so far• Gordon RAM Required– 64GB RAM for Most Steps– 192GB RAM for Assembly• Gordon Disk Required– 8TB for All Subjects– Input, Intermediate and Final ResultsEnabled bya Grant of Timeon Gordon fromSDSC Director Mike NormanVenter Sequencing ofLS Gut Microbiome:230 M Reads101 Bases Per Read23 Billion DNA Bases
Phyla Gut Microbial Abundance Without Viruses:LS, Crohn’s, UC, and Healthy SubjectsCrohn’s UlcerativeColitisHealthyLSToward NoninvasiveMicrobial Ecology DiagnosticsSource: Weizhong Li, UCSD; Calit2 FuturePatient Expedition
We Find Major Shifts in Microbial EcologyBetween Healthy and Two Forms of IBDCollapse ofBacteroidetesExplosion ofProteobacteriaMicrobiome “Dysbiosis”or “Mass Extinction”?On the IBD Spectrum
Almost All Abundant Species (≥1%) in Healthy SubjectsAre Severely Depleted in Larry’s Gut Microbiome
Top 20 Most Abundant Microbial SpeciesIn LS vs. Average Healthy Subject152x765x148x849x483x220x201x522x169xNumber AboveLS Blue Bar is Multipleof LS AbundanceCompared to AverageHealthy AbundancePer SpeciesSource: Sequencing JCVI; Analysis Weizhong Li, UCSDLS December 28, 2011 Stool Sample
Major Changes in LS Microbiome Before and After1 Month Antibiotic & 2 Month Prednisone TherapyReduced 45xReduced 90xTherapy Greatly Reduced Two Phyla,But Massive Reduction in BacteroidetesAnd Large % Proteobacteria RemainSmall ChangesWith No TherapyHow Does One Get Backto a “Healthy” Gut Microbiome?
LS Time Series Gut Microbiome Classesvs. Healthy, Crohn’s, Ulcerative ColitisClassGamma-proteobacteria
Does Intestinal Inflammation Select forPathogenic Strains That Can Induce Further Damage?“Adherent-invasive E. coli (AIEC)are isolated more commonlyfrom the intestinal mucosa ofindividuals with Crohn’s diseasethan from healthy controls.”“Thus, the mechanismsleading to dysbiosis might alsoselect for intestinal colonizationwith more harmful members of theEnterobacteriaceae*—such as AIEC—thereby exacerbating inflammationand interfering with its resolution.”Sebastian E. Winter , et al.,EMBO reports VOL 14, p. 319-327 (2013)E. coli/Shigella Phylogenetic TreeMiquel, et al.PLOS ONE, v. 5, p. 1-16 (2010)*Family Containing E. coliAIEC LF82
DAB1B2ESOur New 2013Reference GenomeSet contains761 Ecoli strains=6x our 2012 SetColored nodesare the 38 strains inthe 2011 PNAS paper
Inflammation Enables Anaerobic Respiration WhichLeads to Phylum-Level Shifts in the Gut MicrobiomeSebastian E. Winter, Christopher A. Lopez & Andreas J. Bäumler,EMBO reports VOL 14, p. 319-327 (2013)
Horizontal Gene Transfer Provides Pathogenic StrainsAdditional Fitness Factors Leading to Growth AdvantageImage from Zhang S., et al. Infect Immun 71: 1–12 (2003)Sebastian E. Winter, Christopher A. Lopez & Andreas J. Bäumler,EMBO reports VOL 14, p. 319-327 (2013)
Does the Gut Microbiome Intermediate BetweenInflammation & the Development of Colorectal Cancer?• Colon Cancer is the most common cancer amongInflammatory Bowel Disease (IBD) patients• IBD is one of the three leading high-risk factors forColon CancerThe root cause of CRC is unclear,but inflammation is a well-recognized risk factor(Wu et al. 2009; McLean et al. 2011)
Fusobacteria Are Found To Be More AbundantIn Colonrectal Carcinoma (CRC) Tissueet al.et al.
LS Time Series Gut Microbiome Classesvs. Healthy, Crohn’s, Ulcerative ColitisClassFusobacteria
Distribution of Relative Species AbundanceAcross the Fusobacteria Phyla in LS001
Class Fusobacteria Is Enrichedin Human Colon Cancer TumorsKostic, A. D., et al. “Genomic analysis identifies association ofFusobacterium with colorectal carcinoma”, v. 22: 292–298 (2012)“…the relative abundance ofFusobacterium was highly enrichedin the population of tumorversus normal samples…”
Could the Presence of Fusobacterium NucleatumBe an Early Indicator of a Transition to CRC?LSCrohn’sFusobacterium nucleatum Relative AbundanceAcross LS, Healthy, UC, and CD
Does Fusobacterium Have a Causal Rolein the Development of Human Colorectal Cancer?“Therefore, our findings of a tumoral enrichmentof Fusobacterium spp. in colorectal carcinomasuggest the possibility thatthese organisms may contribute to tumorigenesis,perhaps in a limited subset of patients,most conceivably by an inflammatory mediated mechanism.”“Our results do not prove a causal relationshipbetween Fusobacterium and colorectal cancer;the establishment or repudiation of such a relationshipwill require further studies of colorectal cancerin both human subjects and animal models of the disease.”Kostic, A. D., et al. “Genomic analysis identifies association ofFusobacterium with colorectal carcinoma”, v. 22: 292–298 (2012)
The Bacterial Driver-Passenger Model for ColorectalCancer InitiationIs Fusobacterium nucleatum a “Driver” or a “Passenger”Tjalsma, et al. Nature Reviews Microbiology v. 10, 575-582 (2012)“Early detection of Colorectal Cancer (CRC)is one of the greatest challenges in the battle against this disease& the establishment of a CRC-associated microbiome risk profilecould aid in the early identification of individualswho are at high risk and require strict surveillance.”
Integrative Personal Omics ProfilingUsing 100x My Quantifying Biomarkers• Michael Snyder,Chair of GenomicsStanford Univ.• Genome 140xCoverage• Blood Tests 20Times in 14 Months– tracked nearly20,000 distincttranscripts codingfor 12,000 genes– measured therelative levels ofmore than 6,000proteins and 1,000metabolites inSnyders bloodCell 148, 1293–1307, March 16, 2012
UCSD Center for Computational Mass SpectrometryBecoming Global MS RepositoryProteoSAFe: Compute-intensivediscovery MS at the click of a buttonMassIVE: repository andidentification platform for allMS data in the worldSource:Nuno Bandeira,Vineet Bafna,Pavel Pevzner,Ingolf Krueger,UCSDproteomics.ucsd.edu
A “Big Data Freeway System” Connecting Usersto Remote Campus Clusters & Scientific InstrumentsPhil Papadopoulos, SDSC, Calit2, PI