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Microbiome Robert Kraaij, PhD Erasmus MC, Internal Medicine - - PowerPoint PPT Presentation

Dec 31, 2022 352 likes •861 views

SNPs and Human Diseases XV November 14th, 2018 Microbiome Robert Kraaij, PhD Erasmus MC, Internal Medicine r.kraaij@erasmusmc.nl Metagenomics - terminology the study of metagenomes genetic material recovered from environmental samples

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SNPs and Human Diseases XV November 14th, 2018

Microbiome

Robert Kraaij, PhD Erasmus MC, Internal Medicine r.kraaij@erasmusmc.nl

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SLIDE 2

Metagenomics - terminology

  • the study of metagenomes
  • genetic material recovered from environmental samples
  • ecological community of microorganisms
  • symbiosis
  • commensal
  • mutual
  • parasitic

OPTION 1:

  • microbiota

 community of microorganisms

  • microbiome

 genomes of the microbiota

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SLIDE 3

Metagenomics - terminology

  • the study of metagenomes
  • genetic material recovered from environmental samples
  • ecological community of microorganisms
  • symbiosis
  • commensal
  • mutual
  • parasitic

OPTION 2:

  • microbiota

 collection of microorganisms

  • metagenome

 genomes of the microbiota

  • microbiome

 community of microorganisms and host

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Microbiota: more than just bacteria…

  • Archaea
  • Bacteria
  • Protozoa
  • Viruses
  • human viruses
  • bacteriophages
  • Fungi
  • molds
  • yeasts
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SLIDE 5

Microbiota: more than just bacteria…

  • Archaea
  • Bacteria
  • Protozoa
  • Viruses
  • human viruses
  • bacteriophages
  • Fungi
  • molds
  • yeasts
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SLIDE 6

The human gut microbiota

  • the forgotten organ
  • 1013 bacterial cells = 1013 body cells
  • ~106 bacterial genes vs ~20,000 human genes
  • many unique functions
  • involved in health and disease!
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SLIDE 7

Density of microbiota increases along GI tract

Walter and Ley (2011) Annu Rev Microbiol.

stool (1011 cells/ml)

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Stool as ‘proxy’ of gut (distal colon) microbiota

collection storage

type 1 type 2 type 3 type 4 type 5 type 6 type 7

profiling metadata

  • Bristol stool scale
  • Rotterdam Study RS-IV
  • n = 836
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Human microbiota: more than just the gut…

urine stool nose tooth eye skin

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SLIDE 10

PHENOTYPE GENOTYPE ENVIRONMENT DIET LIFE-STYLE MICROBIOME

Microbiota

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Gut microbiome and disease associations

  • obesity
  • Crohn’s disease
  • ulcerative colitis
  • eczema
  • asthma
  • diabetes
  • depression
  • etc

hype cycle

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SLIDE 12

Overview

  • Microbiota profiling
  • Data analysis
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Microbiota profiling

  • WHO ARE THEY?
  • WHAT DO THEY DO?
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SLIDE 14
  • culture-based techniques
  • culturomics
  • 16S rRNA marker gene
  • arrays (hitChip)
  • ISpro
  • sequencing
  • microbiome array
  • shotgun sequencing (metagenomics)

Microbiota profiling

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SLIDE 15

IS-proTM profiling

  • 16S-23S interspace (IS region)
  • taxonomy based on size differences

prokaryotic rRNA operon 16S 23S 5S

Bacteroidetes Firmicutes, Actinobacteria, Fusobacteria, Verrucomicrobia Proteobacteria

IS region

FAFV NCBI database

  • 16S – 23S rRNA
  • 8990 entries

Budding et al. (2010) FASEB J.

fragment size (nt) abundance

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SLIDE 16
  • culture-based techniques
  • culturomics
  • 16S rRNA marker gene
  • arrays (hitChip)
  • ISpro
  • sequencing
  • microbiome array
  • shotgun sequencing (metagenomics)

Microbiota profiling

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16S ribosomal RNA gene amplicon

  • highly conserved in bacteria and archaea
  • species-independent PCR amplification
  • variable regions
  • taxonomic classification

16S rRNA

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16S rRNA amplicons

prokaryotic rRNA operon 16S 23S 5S

1500bp  Oxford Nanopore long read sequencing

IS region

~400bp  Illumina MiSeq short read sequencing

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16S RNA analysis pipeline

DNA isolation (NorDiag Arrow) 16S rRNA amplicon Analysis (QIIME) Sequencing (Illumina MiSeq)

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16S rRNA amplicon and sequencing

Fadrosh et al. (2014) Illumina MiSeq

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QIIME-based analysis pipeline Silva database, version 128

Max Planck Institute for Marine Microbiology and Jacobs University, Bremen, Germany

  • September 2016
  • 8,430,487 entries

Read-pair merging Q-score > 19 Chimera filtering Sample QC Reads > mean – 2 SD OTU calling Taxonomy Phylogeny OTU table (anonymous) Biome table (taxonomy) Phylogenetic tree OTU abundancy filtering > 0.005% of total reads Caporaso et al. (2010)

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Read-pair merging

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Chimera filtering

  • chimeras are PCR artifacts
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Chimera filtering

Query Chunk Chunk Chunk Chunk

Ref DB

Hits Query A Query A B normal chimera 4x

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OTU clustering

Operational taxonomic units (OTUs)

  • clustering on basis of homology of the reads (97%)
  • OTUs can be aligned to reference databases
  • unknown OTUs can still be used in analyses
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SLIDE 26

Closed reference calling

  • Each read is compared directly to the database
  • Database determines phylogenetic tree
  • Standardized taxonomy > allows for collaboration
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SLIDE 27
  • culture-based techniques
  • culturomics
  • 16S rRNA marker gene
  • arrays (hitChip)
  • ISpro
  • sequencing
  • microbiome array
  • shotgun sequencing (metagenomics)

Microbiota profiling

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SLIDE 28

Affymetrix Axiom Microbiome array

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SLIDE 29
  • culture-based techniques
  • culturomics
  • 16S rRNA marker gene
  • arrays (hitChip)
  • ISpro
  • sequencing
  • microbiome array
  • shotgun sequencing (metagenomics)

Microbiota profiling

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Shotgun metagenomics

Flaws of 16S rRNA profiling

  • selection introduced by PCR amplification
  • no eukaryotic species such as fungi
  • phylotyping will not give insights into

the gene functions of unknown species

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Shotgun metagenomics

Direct sequencing of DNA

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High output sequencing

  • 2 x 100 bp
  • reads are too short for proper annotation
  • de novo assembly is preferred
  • need for compute power

2 x 100 bp paired-reads de novo assembly ~1 kbp contigs

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Metagenomics technology push

MetaHIT

  • European FP7 project

Human Microbiome Project (HMP)

  • NIH-sponsored project
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Profiling of shotgun data

  • phylotyping databases
  • metagenomic species (MGS)
  • ~7000 MGS specified
  • gene catalogue
  • 8.1 million genes from 760 samples
  • functional databases
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Phylotyping of shotgun data

Arumugam et al., 2011  MetaHIT

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Functional analysis of shotgun data

Arumugam et al., 2011  MetaHIT

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Taxonomic vs functional profiling

  • large taxonomic differences are not reflected in functional profiles

The Human Microbiome Project Consortium (2012)

Samples ordered by taxonomic profiles Samples ordered by functional profiles

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Profiling the gut microbiome

  • WHO ARE THEY?
  • 16S TAXONOMY
  • METAGENOMICS
  • WHAT CAN THEY DO?
  • METAGENOMICS
  • WHAT ARE THEY DOING?
  • METATRANSCRIPTOMICS
  • METAPROTEOMICS
  • WHAT HAVE THEY DONE?
  • METABOLOMICS
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SLIDE 39

Profiling the gut microbiome

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Overview

  • Microbiota profiling
  • Data analysis
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Complex multi-dimensional data

  • no normal or mean profile
  • enterotypes?
  • sparse data
  • many zero abundances
  • limited by technique
  • count data
  • dependent on technique
  • how to normalize?
  • compositional data
  • relative abundances add up to 1
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Diversities

  • α-diversity
  • diversity within a sample
  • biological metric
  • number of species * evenness
  • β-diversity
  • diversity (distance or dissimilarity) between samples
  • UniFrac distances
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OTU table

OTU id sample_01 sample_02 sample_03 sample_04 … OTU_12 3 456 343 OTU_318 34 45 3 2 OTU_37 567 2134 478 675 … … … … … … Total 5,975 4,952 6,735 5,374

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Rotterdam 16S rRNA datasets

Domain Phylum Class Order Family Genus OTUs (2) (11) (18) (24) (43) (183) (777) Class Domain Phylum Order Family Genus OTUs (1) (7) (15) (19) (36) (152) (661)

Shannon Diversity Index Shannon Diversity Index

5 major phyla

N=2,111

N=156

N=1,427

N=1,135 N=1,106

Generation R Study 9-11 year-olds Rotterdam Study adults

Radjabzadeh et al. (2018) in preparation

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SLIDE 45

Children vs adults

  • Generation R Study vs Rotterdam Study

N=2,111 N=1,427 GenR RS *** 8 7 6 5 4 3 2 Shannon diversity index

average phylum-level profiles

Radjabzadeh et al. (2018) in preparation

Shannon alpha diversity

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SLIDE 46

MiBioGen consortium

  • Meta-analyses of gut microbiome GWAS
  • > 20 cohorts (still including)
  • > 20,000 samples
  • 16S rRNA profiling (Illumina)
  • 226 genera
  • 8M HRC1.1 imputed SNPs

NGRC

Traits

  • Shannon alpha-diversity
  • Binary trait (presence/absence)
  • Quantitative trait (abundance)
  • Beta-diversity
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MiBioGen consortium

Cohort name Population 16S domain Genotyping method N Description

1

LLD Netherlands (Caucasian) V4 Illumina Immunochip, Cytochip 1089 Representative of population

2

NGRC Netherlands (Caucasian) V1-V2 PsychChip (Broad Institute, Boston, USA) 153 Healthy group + ADHD group

3

RS Netherlands (Caucasian) V3-V4 Illumina 550k 1427 Representative of population

4

GENR Netherlands (multi-ethnic) V3-V4 Illumina 610k 2111 Representative of population

5

NTR Netherlands (Caucasian) V4 Affymetrix 6.0 499 Twins

6

MIBS_Co Netherlands (Caucasian) V4 Illumina OmniExpressExome 111 Healthy volunteers

7

FGFP Belgium (Caucasian) V4 Illumina OmniExpress 2482 Representative of population

8

SHIP Germany (Caucasian) V1-V2 Affymetrix 6.0, Illumina OmniExpressExome, Exomechip 1904 Representative of population

9

SHIP-TREND Germany (Caucasian) V1-V2 Affymetrix 6.0, Illumina OmniExpressExome, Exomechip (-) Representative of population

10

FOCUS Germany (Caucasian) V1-V2 Illumina Immunochip, Exome 1555 Representative of population

11

BSPSPC Germany (Caucasian) V1-V2 Illumina 550K, Immunochip, Metabochip, Affymetrix 6.0, Axiom 912 Representative of population

12

TwinsUK UK (Caucasian) V4 HumanHap300, Hap610Q, 1M-Duo, 1.2M-Duo 1793 Twins

13

CHRIS Italy (Caucasian) ? ? ? ?

14

COPSAC Denmark (Caucasian) V4 Illumina OmniExpress 424 Representative of population

15

POPCOL Sweden (Caucasian) V1-V2 Illumina MiSeq 250 Representative of population

16

METSIM Finland (Caucasian) V4 Illumina OmniExpressExome 531 Representative of population

17

PNP Israel (Israeli) V3-V4 Metabolochip 1066 Healthy volunteers

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GEM_HCE_v12 Canada, USA, Israel (Caucasian, Israeli) V4 Illumina HumanCoreExome, Immunochip (-) Healthy individuals

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GEM_HCE_v24 Canada, USA, Israel (Caucasian, Israeli) V4 Illumina HumanCoreExome, Immunochip (-) Healthy individuals

20 GEM_ICHIP_HCE

Canada, USA, Israel (Caucasian, Israeli) V4 Illumina HumanCoreExome, Immunochip 1543 Healthy individuals

21

CARDIA USA (Caucasian and African-American) V3-V4 Illumina Exome, Affymetrix 6.0 282 Representative of population

22

HCHS/SOL USA (Hispanics/Latinos) V4 Illumina Omni Chip + custom array 1778 Representative of population

23

KSCS Korea (Asian) V3-V4 Illumina HumanCore BeadChips 12v 833 Representative of population

23 cohorts >21,000 samples

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MiBioGen consortium

55 bacterial taxa (1,232 SNPs)

  • GWAS quantitative trait
  • 226 genera
  • 8M SNPs
  • Meta-analysis

MiBioGen consortium (2018) unpublished

LCT locus

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SLIDE 49

Questions…

? ?