013 luna 2012 beaumont dna final

7/28/2019 013 Luna 2012 Beaumont DNA Final

1/10

9/25/20

RuthAnnLuna,PhD,MB(ASCP)CM

TexasChildrensMicrobiomeCenter

TexasChildrensHospitalHouston,TX

BacterialIdentification Single

Pathogen

Identification

by

16S

rRNA

Sequencing WhatisaMicrobiome? NextGenerationSequencingof16SrRNA

CurrentMicrobiomeResearch MicrobiomeCharacterizationasaDiagnostic

ConventionalMicrobiology

Culturedriventechniques

Biochemicaltesting(manualandautomated)

MolecularMethodsforDirectDetection

Singleplexandmultiplexamplificationbasedassays

Speciesspecificsequencing

ConventionalMicrobiology+Molecular

Intheearly2000s,difficulttoidentifyorganismswerecommonlyreportedwithgenericlanguagesuchasgramnegativerod,unabletofurtheridentify.

Theseproblemorganismshistoricallyyieldedconflictingresultsbetweenanarrayofidentificationtechniquesincludingmorphology,gramstains,biochemicaltesting,andoftentheopinionofaseasonedmicrobiologist.

Ledtothedevelopmentofasequencingbasedclinicalassayforbacterialidentification.

Approximately1542nucleotidesinlength

Welldocumentedseriesofboth

highlyconserved and

variable

regions Universalprimers target

conservedregions,theoreticallyamplifyinganybacterium.

Variableregions canbeutilizedtodifferentiatebetweenbacterialorganisms.

http://www.biochem.umd.edu/biochem/kahn/

The16SrRNA geneiscommonlytargetedbyrealtimePCRandsequencingassaysfortheidentificationofbacterialpathogens.

Thoroughanalysis

of

regions

V1

V8

highlighted

thediscriminatorypowerofregionsV2,V3,andV6withV1proventobeespeciallyusefulinStaphylococcussp.(Chakravorty,etal.2007).

V1 V2 V3 V4 V5 V6 V7 V8 V9C1 C2 C3 C4 C5 C6 C7 C8 C9

1 1542


2/10

9/25/20

V1

~115 bp amplicon


V6

~100 bp amplicon

1 1542

V3

~190 bp amplicon

16S rRNA

1542 bp

V6AmpliconV1Amplicon

V1R

V1F

V6F

V6R

6 120 965 1065

V1Amplicon(115bp) Amplificationprimers

Sequencingprimers

V6Amplicon(100bp)

B

B

UniversalprimersadaptedfromJonasson,etal.APMIS2002.

V3Amplicon(193bp) Amplificationprimers

Sequencingprimer

16S rRNA

1542 bpV3Amplicon

V3F V3R

341 533

B

16S rRNA

1542 bp


V1R

V1F

V6F

V6R

6 120 965 1065

V3Amplicon

V3F V3R

341 533

Datafromallthreeregionsprovidedanaverageof109bppersample.

Ingeneral,V3andV6providedthemostreliable

and

useful

data. However,V1remainsimportantforcertaingenera.

Bacterial DNA extraction

Pyrosequencing

Sequence analysis and final

identification

Amplification of V1, V3, & V6

variable regions of 16S rRNA


3/10


4/10


5/10

9/25/20


16S rRNA1542 bp

V1V3Amplicon

507bp

V1V3RV1V3FV6V9F

V6V9R

27 534 968 1492

V3V5F V3V5R

357 926

V3V5Amplicon

569bp

V6V9Amplicon

524bp


V1R

V1F

V6F

V6R

6 120 965 1065

V3Amplicon

V3F V3R

341 533

SubjectRecruitment/Enrollment&SpecimenCollection

ClinicalCollaborators

NucleicAcidExtraction

TCMCCoreStaff

ClinicalMetadataCollection

TCMCResearchCoordinator

NextGenerationSequencing

TCMCCoreStaff

DataAnalysis

TCMCBioinformatics

Multipleplatformscapableofgeneratingalargenumberofsequencingreadsperrun/sample ABISOLiD

Illumina

IonTorrent

PacificBiosciences

Roche454 Sequencelengthanddepthvariesacross

platforms.

TCMCcurrently

utilizes

aRoche

454

platform.

NucleicAcidExtraction

CommercialManualExtractionKits

EmulsionPCR

OneBead=OneRead

Amplification

DesiredSingleorMultipleTargets

Sequencing

Sequencing bysynthesis

Data

AnalysisParsingsequenceresultsbasedonuniqueidentifiersattachedtoeachsequence

ConventionalPCRamplification

DNAinputof~20ng

Eachsample

amplified

individuallytargetingregionofinterest

UtilizinglowDNA

reagentswhen

possible

454Primer Providescommonsequencefor

nextamplification

step

Key QCindicatorforsequencingphase MID Molecularidentifier;barcodespecificto

anindividualsample TargetspecificPrimer Ex.16SrRNA variable

region

454Primer Key MID TargetspecificPrimer


6/10

9/25/20

Amplicons fromtheinitialPCRreactionarehybridizedwithDNACaptureBeads.

Eachbead

carries

aunique

single

stranded

library

fragment. Beadsareemulsifiedwithamplificationreagentsina

waterinoilmixturetotrapindividualbeadsinamplificationmicroreactors.

Entireemulsionamplifiedinparalleltocreate

millions

of

clonal copies

of

each

library

fragmentoneachbead. Emulsionisthenbrokenwhiletheamplified

fragmentsremainboundtotheirspecificbeads.

Amplificationtargets

the454Primerthat

waslocatedonthe5endofthelibraryconstructionprimer.

BeadsareloadedontothePicoTiterPlate(PTP)device ThesurfacedesignofthePTPallowsforonlyonebead

perwell. PTPDevicecanbedividedinto2,4,8,or16regions. PTPDeviceisloadeddirectlyintothesequencing

instrument.

Individualnucleotidesflowedinsequenceacrossthewells.

Eachincorporationofanucleotidecomplementarytothetemplatestrandresultsinachemiluminescent lightsignalrecordedbythecamera.

~2millionwellsontheplate

Signalintensityofeachincorporationeventateachwellpositiondeterminesthesequenceofallreadsinparallel.

Heightofeachpeakrepresentssignalintensity, which

translatesinto

number

of

nucleotides

at

that

position

(ex.2Asor3As) Lackofpeakindicatesnoincorporation

DNAExtraction

DNAisolationfrommultiplebodysites

dictatedbystudydesign

Amplification

Targetingconserved

and

neighboring

variableregionsofthe16SrRNA gene

454AmpliconDirectedSequencing

Sequencing ofmultipletargetsforbacterial

identification

DataAnalysis

Identificationofdiseasesignaturesbasedonshiftsinthemicrobiome

V1 V2 V3 V4 V5 V6 V7 V8 V9

1 1542

http://www.biochem.umd.edu/biochem/kahn/


7/10

9/25/20

Organismsmustcompetetobeisolatedfrom

initial

culture

(low

abundance

organisms

will

bemissed). Duetotimeandcost,alimitednumberof

growthconditionsareroutinelyutilized. Unculturable organisms unknownpreferred

growthconditionsornovelorganisms Focusisonidentifyingpathogenicorganisms

ratherthanassessingthecompletebacterialcommunity.

AggregateData

Removechimeric sequences

QualityFilter

Assessmatch/mismatch

to

MIDs,

primers

Removelowquality/ambiguousbasecalls

Removesequencesthataretooshort/long

Minimizesequencingnoise

IdentifyOTUs

Compareamong/betweencommunities

AssignidentitiestoOTUs/sequences

Putanameonitdefiningoperationaltaxonomicunits(OTUs)

Numericalspecies

16SrRNAgenes,sequencessharing>97%similarity

Applicationofclassicalecologicalmetrics

Comparingcompositionamongcommunities

Sharedvs.uniquetaxa

Differingproportionsofthosetaxa

Measuringdiversity Alphadiversity(within

sample/subject/group)

Betadiversity(between

samples/subjects/group)

Baselineofhealthyadults

18differentbodysitessampled

Standardizationof

Specimencollection

Processing

Sequencing

Analysis

CHuttenhoweretal.Nature486,207214 (2012)doi:10.1038/nature11234 CHuttenhoweretal. Nature486,207214 (2012)doi:10.1038/nature11234


8/10

9/25/20

CHuttenhoweretal. Nature486,207214 (2012)doi:10.1038/nature11234

HumanSkinSitesSurvey

Grice,etal,Science 2009

Gastroenterology

Irritablebowelsyndrome(IBS)

ShortBowelSyndrome(SBS)

Neonatology

Prematureinfants(longitudinalanalysisplusdietcomparisons)

Developmentofbronchopulmonarydysplasia

Pulmonary

Cysticfibrosis

(CF)

ChildrenwithIBS(N=23subjects)

Bacteroidetes

Firmicutes

Verrucomicrobia

Proteobacteria

Actinobacteria

Fusobacteria

Spirochaetes

TM7

Cyanobacteria

Synergistetes

Euryarchaeota

Healthy adults (N=48 subjects)

Adults

Healthychildren(N=22subjects)

Bacteroidetes

Firmicutes

Verrucomicrobia

Proteobacteria

Actinobacteria

Fusobacteria

Spirochaetes

TM7

Cyanobacteria

Synergistetes

Euryarchaeota

PhylaDistributionsinChildrenandAdults

GreaterAbundanceofGammaproteobacteriaFoundinGut

MicrobiomesofChildrenwithIBS

#Significantlydifferent(p


9/10

9/25/20

SBSisdefinedasinsufficientsmallintestinelengthtomaintainproteinenergy,fluid,electrolyteor

micronutrient

balances

when

onaconventionallyaccepted,normaldiet.

Pediatric causesincludegastroschisis ,intestinalatresias,andnecrotizingenterocolitis

SBSischaracterizedbyrecurrentdiarrheaandintestinalmalabsorption.

Clinicaloutcomesareinfluencedbyage,etiologyofSBS,lengthofremainingbowel,incidenceofsepsisandliverdisease,andsuccessfuldietmodifications.

Davidovics,2012(submitted)

Weeks12

(blue)

Weeks36

(green)

Luna,2012(inpreparation)

Ordercompositionbyweekoflife Changes in the bacterial genera present in the respiratory micr obiomeof patients with CF from 1 month to 9-18 months of age.

Madan J C et al. mBio 2012; doi:10.1128/mBio.00251-12

RecentstudiesreportingthatroutinelyidentifiedpathogensinCFwerenotpresentinsignificantabundancebasedonnextgenerationsequencingdata.

PilotstudyattheTCMCconfirmsthisscenario. DatatobepresentedattheAssociationforMolecular

PathologyAnnual

Meeting

next

month

(platform

and

posterpresentation) 35differentgeneraidentified(rangeof415generaper

specimen)

MostprevalentgeneraincludedStreptococcus,Prevotella andVeillonella

Culturefindingswerenotalwaysidentifiedinnextgenerationsequencingresults

Cultureidentifiedpathogensfoundatvaryinglevelsofrelativeabundanceintheoverallbacterialpopulation.

Establishingbaselines HMP healthycomparisondataset

Diseaseonsetforlongitudinalassessments

Geographicalordemographicgroupsubsets Diseasestatecomparisons Healthyvs.disease

Acrosssimilardiseases Translationalresearchedgingclosertothe

clinicalrealm


10/10

9/25/20

Microbiomecharacterizationwillevolveintoa

clinicaldiagnostic

tool.

Initialscreeningforunknowninfections

Companiondiagnosticforavarietyofscenarios

Preandpostinterventionassessment Considerationintreatmentselection Accumulationofmicrobiome datainaspecific

populationcouldalterrecommendedtreatmentpaths Impactonpatientprognosis Possibilityofpredictivecriteriabasedonsequential

microbiome evaluations(ex.successoftreatmentoroddsofrecurrence).

Gramstain

CultureonCCFAAgar

Texas Childrens Microbiome Center

James Versalovic, MD, PhD

Emily Hollister, PhD

Jennifer Spinler, PhD

Toni-Ann Mistretta, PhD

Jessica Runge

Yue Shang

Michelle Rubio-Gonzales

Sabeen Raza

BCM and TCH Pedi GI group

Robert Shulman, MD

Bruno Chumpitazi, MD

Erica Baimbridge

Alkek Center for Metagenomics and

Microbiome Research and Human

Genome Sequencing Center

Richard Gibbs, PhD

Joseph Petrosino, PhD

Bioinformatics Research Lab

Aleksandar Milosavljevic, PhD

Kevin Riehle, MS

Christian Coarfa, PhD

Thank you to the

Children and theirFamilies

ASpecialThankYoutotheNIHCommonFundandtheNationalInstituteofDiabetes,Digestive

andKidneyDiseases(NIDDK)

013 luna 2012 beaumont dna final

Documents