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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
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V1
~115 bp amplicon
V1 V2 V3 V4 V5 V6 V7 V8 V9C1 C2 C3 C4 C5 C6 C7 C8 C9
V6
~100 bp amplicon
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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
V6AmpliconV1Amplicon
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
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V1 V2 V3 V4 V5 V6 V7 V8 V9C1 C2 C3 C4 C5 C6 C7 C8 C9
16S rRNA1542 bp
V1V3Amplicon
507bp
V1V3RV1V3FV6V9F
V6V9R
27 534 968 1492
V3V5F V3V5R
357 926
V3V5Amplicon
569bp
V6V9Amplicon
524bp
V6AmpliconV1Amplicon
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
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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
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http://www.biochem.umd.edu/biochem/kahn/
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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
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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
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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
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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)