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MICROBIOMICS –Current and future tools of the trade
Ingeborg KlymiukCore Facility Molecular BiologyZMF - CENTER FOR MEDICAL RESEARCHMedical University Graz
MICROBIOMICS OMIC technologies make up a holistic view of the molecules that make up a cell, tissue, sample or
organism OMICS: universal detection of genes (genomics), mRNA (transcriptomics), proteins (proteomics),
lipids (lipidomics) and metabolites (metabolomics) in a specific biological sample Non-target, non-biased manner (bias from study design to analysis that can impact results) approaches for biomarker discovery
D E F I N I T I ON O F O M I C T E C H N O L O G I E S
ProteOMICS
LipidOMICS
MetabolOMICS
TranscriptOMICS
MetagenOMICS
culturOMICsselective growthantibiotic assayenzymatic assay
X
AYTargeted amplicon
16s amplicon NGSLEA-Seq
PhyloChips
Conduction a microbiome study
The approaches to study the human-associated microbial
communities are increasing
DNA or RNA based analysis
community surveys: (descriptive) indentification of microorganism
(OTUs - operational taxonomical units); differences in OTU
composition between sample groups
indentification of genes: functional identification of genetic
potential, gene richness
detection of rare OTUs, minor species: depth requirements
transcriptional activity and functionality
various constituents of a microbial community, such as
eukaryotes, viruses and various groups of bacteria
live dead discrimination: propidium monoazide (PMA)
duration, costs and sample volume of analysis
Goodrich et al. Cell 158, July 17, 2014
biological question
Study subjects and controls
SamplingSample storage
NS extraction (DNA, RNA)PCR, libraray preparation
sequencing
pipeline specific analysisdiversity analysis
classification, clustering, modeling
OMICS data analysis
Deposit data, share…
Conduction a microbiome study
16s amplicon basedapproach
Who is there?
descriptive view ofmicrobiome diversity
assess the generalcomposition of the
microbiota
economical andtherefore scale to large
projects
bacterial, archaeal, fungal diversity
complex bacterialcommunities
Metagenomics
What can they do?
portrays functionalpotential of microbiome
gene content
bacteria, archaea, fungi, viruses
human/host background
Metatranscriptomics
What are they doing?
describes active geneexpression
elucidates the activemembers
bacteria, archaea, fungi, viruses
human/host background
(16s) targeted amplicon
one or a few marker genes and use these markers to reveal the composition and diversity of themicrobiota
16s rRNA gene highly conserved between different species of Bacteria and Archaea the internal transcribed spacer (ITS) region of the rRNA – fungi (Bellemain et al. 10, Bokulich et al. 13) beside conserved primer binding regions hypervariable regions provide species-specific signature
sequences primer choise, variable region, effect of experimental setup: PCR amplification, -cycle number, -
condition, depth of analysis, platform used fro sequencing …..effects on results large databases of reference sequences and taxonomies (such as greengenes - DeSantis et al.06,
SILVA - Quast et al. 13 and the Ribosomal Database Project - Cole et al. 09) risk of misclassification
(16s) targeted amplicon workflow
DNA Isolation
amplicon preparation
indexing, purificationand pooling
sequencing
1 100 200 300
(16s) targeted amplicon workflow DATA ANALYSIS
Galaxy: mothur, qiime
(optional) combine two sets of reads
Quality filtering and trimming
Pick OTUs with uclust (similarity 0.97)
Taxonomy assignment
Representative sequence alignment
PCR bias, Chimera removal (chimera.uchime)
Phylogenetic classification
Alpha diversity (Chao, shannon, eveness, richness)
Beta diversity (UniFrac, Bray-Curtis, Euclidian, Pearson)
Diversity analysis, sample and group comparison of
microbial communities (multi-variant data
analysis)Statistics and visualization (multi variant data
analysis)
Kozich JJ, Westcott SL, Baxter NT, Highlander SK, Schloss PD. (2013): Development of a dual-index sequencing strategy and curationpipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. Applied and Environmental Microbiology.79(17):5112-20.
Goodrich et al. Cell 158, July 17, 2014
innovations for high-throughput amplicon sequencing PCR and sequencing introduce sequence errors and sampling bias poor estimates of microbial
diversity the amplification of non-target DNA may results in inefficiencies to represent the microbial pattern low diversity samples are problematic on the Illumina system
1) increase amplicon diverisity by spike sequencing runs with shared genomic DNA (PhiX174)2) increase diversity by heterogenity spacer, frameshift nucleotides3) low-error amplicon sequencing: LEA-Seq…..
Lundberg et al nature methods 2013 Faith et al Science 2013 Fadrosh et al Microbiome, 2014
PhyloChip G3
PhyloChip G3 is a microbial community assessment tool that can simultaneously track high-abundance and low-abundance bacterial and archaeal taxa; currently available through the SecondGenome Inc. (San Francisco, CA)
Microarrays based technology with a high chip-to-chip reproducibility
16s full length ampliconBacteria: 27F 5‘-agagtttgatcctggctcag-3‘, 1492R 5’-ggttaccttgttacgactt-3’Archaea: 4Fa 5‘-tccggttgatcctgcccg-3‘, 1492R 5‘-ggttaccttgttacgactt-3’
PhyloChip G3
PhyloChip G3 25-mer oligos, 1,100,000 probes and analysis for 59,959 OTUs overcome the depth bias - no saturation effect detection of rare OTUs; down to subspecies level resolution Mainly used for environmental samples (space crafts, clean rooms, hot pring systems,…) Human high diversity samples, gut samples, high resolution and sensitivity desired ‚tackling the minority‘ Used in varius publications characterizing thediseased human intestinal tract, gastric samples,…
Moissl-Eichinger et al 2014
MetagenOMICS
the ‘unbiased’ direct sequencing of the microbiomes, genomes of all microorgansims in a sample
1014 microorganisms inhabiting the human gut
ensemble of the genomes of human-associated microorganisms
providing much richer data on the functional potential present in microbial community genomes
sacrifice resolution
Extend the analysis to other organisms than bacteria
MetaHIT: defined a gene catalogue of 3.3Mio non redundant gut microbial genes by metagenomics
defining 100 times more genes than encoded by the human host genome
DNA Isolation
libraray preparation
indexing, purificationand pooling
sequencing
MetagenOMICS - workflow
Reference based: MIRA, Newbler
De novo: SOAP, Velvet
MG-RAST, a metagenome annotation system
MetagenOMICS - workflow
sequencing
assembly
Annotation, gene prediction
Binning
Statistical analysis
Data storage
Data sharing
Metadata
16s phylogeny
QCQC
draft genome
MetagenOMICS
integrating microbial membership with biomolecular potential and activity in the human intestine eliminates danger of PCR/Primer bias (missed phyla) Genomes form the same species (by 16s) can have large genomic differences outside the 16s
region, can have different sets of gene clusters includes fungal (<1% stool) and viral (1*10-5% – 2% stool) sequences; immense biological
function (reviewed in Morgan et al Gastroenterology 2014)
Lepage et al Gut 2013 Dutilh et al Nature 2014
MetatranscriptOMICS
only a subset of the present genes are expressed
rare species might be highly transcriptional active
characterize the complete collection of transcribed sequences in
a microbial community
How communities respond to changes in their environment
Analysis of the active fraction of the community
Is a gene active?
Is a gene higherexpressed than another
gene in the same sample/treatment
Is a gene differentiallyexpressed in response toexperimental conditions?
Does gene expressionchange over time?
MetatranscriptOMICS-workflow
RNA is less stablerRNA, tRNA deplition (stool: 98%) (MicrobExpress kit; LifeTech)mammalian (host cell) RNA selectively removed; high relevance for e.g.
biopsy samplesHigher amount of starting material is necessary or amplification step
mod. from Warnecke et al 2009
Total RNA extraction
Microbial Community
mRNA enrichment
cDNA synthesis
optional: amplification
Libraray preparationfor high throughput
sequencing
Metatranscriptome
Franzosa et al 2014 PNAS
de-novo Sequencing
Amplicon Sequencing
Transcriptomics (e.g. Tag sequencing, RNA Seq)
ChIP-Sequencing
meDIP-Sequencing
Haplotyping
Microbiome Studies/Metagenomics
Transcriptome-wide full-length cDNA Sequencing
Whole Genome Re-Sequencing
Exome Sequencing
read length
read
num
bers
ncRNA transcriptomics
Roche 454GS FLX
1.3Mio reads á 700b
Illumina HiSeq2000/2500
~2bil. reads á 2x125b
Pac. Biosciences
50k reads 5/8.5kb-20kb!
Illumina MiSeq
~25Mio. reads á 2x300bp
Illumina NextSeq500
~400Mio. reads á 2x1500bp
Which Technology/System to choose?
Illumina: sequencing by synthesis
Output scalable: MiSeq, HiSeq, NextSeq High multiplexing capacity Read length increasing (300bp MiSeq,150 HiSeq) Low error rates Cost efficient system (about 1400€ for a MiSeq run-384
samples)
Pacific Bioscience: SMRT Cell
www.pacificbiosciences.com
small genome, bacteria, archaea metagenomics no multiplexing capacity One SMRT cell 200-400MB Output
Nanopores – The future in DNA sequencing
Single moelcule sequencing Direct RNA molecule sequencing High error rate (13%-15%)
MALDI-TOF MS
ProteOMICS
mass spectrometriy techniques offer methods to directly analyze small molecules determine lipids, metabolites and proteins MALDI-TOF: routine application for bacterial classification (DSMZ) recording of mass spectra of large biomolecules (mainly ribosomal proteins) mass spectrometric fingerprints: identification of bacteria, yeasts and fungi by
comparison with reference databases
http://www.mayomedicallaboratories.com/articles/communique/2013/01-maldi-tof-mass-spectrometry/index.html
LipidOMICS / MetabolOMICS
Metabolomics and metabolome profiling for disease biomarkers
Understand rare taxa and taxa with genomic variations
Rare taxa can have important metabolic activities
Metabolomics provide o picture of metabolism rather than the potential of metabolism
short-chain fatty acids (SCFAs) derived from microbial metabolism in the gut play a central role in host homeostasis
Ursell et al. Gastroenterology 2014
RT: 2,68 - 18,86
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18Time (min)
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9,014,25 6,73 7,36 8,32 17,479,73 11,12 14,16 16,57 17,9014,8311,66 12,15 14,08 16,31
NL:2,28E7TIC F: + c Full ms [80,00-400,00] MS L12
Semi - Targeted Methods16:0
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MetabolOMICS
CulturOMICS
it is commonly accepted that c. 80% of the bacterial
species found by molecular tools e.g. in the human gut
are uncultured or even unculturable (Turnbaugh et al
2007)
the German national academy Leopoldina in Berlin has
recently recommended increasing the effort in
taxonomic research
taxonomy is important for medicine, food technology
and agriculture, for an optimal understanding and
application of microorganisms
pure cultures are mandatory for taxonomic assessment
CulturOMICS is an approach allowing extensive
assessment of the microbial composition by high-
throughput culture
complements the metagenomic analysis and
overcomes the ‘depth bias’ and DNA isolation/PCR bias
CulturOMICS
define high throughput culture conditions (212 different )
high throughput automized colony screening
indentification by MALDI-TOF
compariosn of culturomics taxa with those found by 16s amplicon sequencing
31 new bacterial species and genera (http://www.ebi.ac.uk/embl/Submission/index.html)
culturomics approach yielded 340 bacterial species, seven phyla, 117 genera
pyrosequencing identified 282 species, six phyla, 91 genera
51 phylotypes overlap between the methods
definition of most efficient culture conditions
characteriation by functionality and enzyme activity possible
minority population can have a substantial effect on the ecology of the gut
microbiota and on human health
later use of indetified sepcies as probiotica
Challenge & impact of OMICs
Owyang & Wu 2014
Proteomics Genomics
Hyb
Amplicon
Metagenome
Transcriptome
LipidomicsMetabolomics
Bioinformatics
candidate list
Omics
• Functional analysis • Candidate prioritization• Biomarker identification• Drug target discovery
Integrative Bioinformatics Analysis & Methods
Reiss et al. 2011, Host Cell & Microbe
Challenge & impact of OMICs
THANK YOU!
Ingeborg KlymiukCore Facility Molecular BiologyZMF - CENTER FOR MEDICAL RESEARCHMedical University Graz
Advantage Disadvantage
Present in all bacteria and archaea (91).
Present in multiple copy numbers throughmost organisms (91), which may lead tooverestimation of the abundance of someorganisms.
Contains highly conserved regions suitablefor universal primer design (37).
Small number of organisms do not display asmuch conservation through these regionsleading to primer bias (37).
Contains regions of high variability suitableas unique identifiers (42).
Regions of variability are occasionallyinsufficient to provide species-levelresolution, and may be biased toward certainspecies (39,42).
Numerous well-curated databases allowingsequence comparison and taxonomicassignment of organisms (45).
Many databases contain sequences witherrors (45).
Well-studied primer pairs available, whichare capable of amplifying most organismswith high specificity for bacteria (38).
May lack specificity for certain bacterialgroups and result in inaccurate estimations ofcommunity composition (38).
16s pros and cons
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