Transcript 2016_Huttenhower_Stamps05

Functional profiling with HUMAnN2
Curtis Huttenhower
Galeb Abu-Ali
Eric Franzosa
Harvard T.H. Chan School of Public Health
Department of Biostatistics
08-12-16
The two big questions of microbial
community analysis...
Who is
there?
What
are they
doing?
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Metagenomic analyses:
molecular functions and biological roles
Orthology:
Structure:
Biological roles:
Grouping genes by conserved
sequence features
Grouping genes by similar
protein domains
Grouping genes by pathway
and process involvement
COG, KO, FIGfam…
Pfam, TIGRfam, SMART, EC…
GO, KEGG, MetaCyc, SEED…
Warnecke, 2007
Turnbaugh, 2009
DeLong, 2006
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← Phylum abundance →
← Phylum abundance →
“Who’s there,” versus, “What they’re doing,”
in the healthy human microbiome
← Subjects →
Oral (BM)
http://hmpdacc.org/HMMRC
Oral (SupP)
Oral (TD)
Gut
Vaginal
← Pathway abundance →
Skin
← Pathway abundance →
Nares
← Subjects →
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HUMAnN2: Organism-specific functional profiling
of metagenomes and metatranscriptomes
Eric
Lauren
Franzosa McIver
http://huttenhower.sph.harvard.edu/humann2
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HUMAnN2: stratified output
UniRef gene cluster
Gene name
Total gene abundance (RPK)
UniRef90_R6K3Z5: IMP dehydrogenase
600.95
UniRef90_R6K3Z5: IMP dehydrogenase|Bacteroides_caccae
234.76
UniRef90_R6K3Z5: IMP dehydrogenase|Bacteroides_dorei
107.38
UniRef90_R6K3Z5: IMP dehydrogenase|Bacteroides_ovatus
92.18
UniRef90_R6K3Z5: IMP dehydrogenase|Bacteroides_stercoris
83.95
UniRef90_R6K3Z5: IMP dehydrogenase|Bacteroides_vulgatus
57.27
UniRef90_R6K3Z5: IMP dehydrogenase|unclassified
25.41
Σ
Per-species & unclassified stratifications
MetaCyc pathway
Pathway abundance & coverage
PWY-7221: GTP biosynthesis
200.35
1
PWY-7221: GTP biosynthesis|Bacteroides_caccae
120.23
1
PWY-7221: GTP biosynthesis|Bacteroides_dorei
11.12
0
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HUMAnN2 synthetic evaluation (genes)
…and is >7x faster
~2.3 hours
~0.3 hours
HUMAnN2 tiered search is
more accurate…
Comprehensive search
suffers from spurious hits
Compare exp. vs. obs.
gene abundance
1x
Staggered abundance
~0.1x to 100x coverage
Synthetic human gut
metagenome (top 20 species)
(10M reads,
8 cores)
...and
provides
accurate
per-species
quantification!
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The other HMP2: HMP1-II
Tongue
Dorsum
Oral
2012
Ns
Nt
2016
403 1248
44
153
Skin
2012
Ns
Nt
2016
118 306
18
64
2012
Ns
Nt
Ns
Nt
2012
Ns
Nt
159
Urogenital
2012
2016
Ns
68
183
Nt
17
54
2016
121 378
35
2012
Ns
Nt
2016
117 370
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• Shotgun metagenomes
• 18 body sites in 5 areas
 6 intensively sampled sites
• 2,285 samples from 264 people
• Up to 3 time points per (person, site)
114
Buccal
Mucosa
2016
152 548
49
133 413
41 116
Supragingival
Plaque
Gut
2012
2016
105
• New analyses:
 HUMAnN2: Enzyme and pathway
abundances, stratified by species
 MetaPhlAn2: Eukaryotes and viruses
 StrainPhlAn: Strain profiling
 Temporal dynamics
 Fully updated IDBA-UD assemblies +
gene catalog / annotations
HUMAnN2 real-world performance
~60% of reads align before
translated search
~15% more reads align during
translated search (total~75%)
Applied HUMAnN2’s tiered
search to profile 100s of
human metagenomes
(HMP, six major body sites)
Pangenome search tier
1-2 orders of magnitude faster
than comprehensive
translated search
DIAMOND w/
comprehensive
protein db
bowtie2 w/
sample-specific
pangenome db
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HUMAnN2 identifies body site-specific
“signature pathways” in the human microbiome
Zoom in…
Max area
≈2%
relative
abundance
(other areas
square-root
scaled)
• “signature for area i” → Q1( area i ) > Q3( area j ) for all j ≠ i; very stringent!
• ≈50 total signature pathways across 4 major body areas (20 shown)
• Values plotted = median (Q2) abundance for samples from that area
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HUMAnN2 identifies body site-specific
“signature pathways” in the human microbiome
Zoom in…
Unclassified abundance
In the minority
L-rhamnose degradation (RHAMCAT-PWY)
is a signature of the human gut microbiome
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HUMAnN2 reveals three distinct mechanisms
of cross-environment functional conservation
Mechanism 1: Complex
Multiple contributing species per individual
Ex: L-rhamnose degradation in gut microbiome
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HUMAnN2 reveals three distinct mechanisms
of cross-environment functional conservation
Mechanism 2: Per-person-dominant
One dominant contributing species per individual
Ex: peptidoglycan biosynthesis in vaginal microbiome
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HUMAnN2 reveals three distinct mechanisms
of cross-environment functional conservation
Mechanism 3: Universal-dominant
Same, dominant contributing species in all individuals
Ex: trehalose degradation in skin microbiome
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HUMAnN2 reveals unusual “relative expression” in
paired metatranscriptomes & metagenomes
Sucrose degradation follows a complex attribution
pattern across ~200 human gut metagenomes…
In collaboration with
…but its expression can be dominated by a single
the STARR Consortium
species in paired gut metatranscriptomes!
& HPFS cohort
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Conclusions
HUMAnN2 implements a
tiered approach to faciliate
meta’omic functional profiling
This approach is more accurate
& much faster than traditional
comprehensive meta’omic search
Results stratify by species for free:
Answering “who’s there?” and
“What are they doing?” in tandem
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Further improving short reads and regions of
local homology among proteins
• Protein of interest
• Belongs to a family
• Local homology to
unrelated families
• Short reads from
unrelated families may
map to protein of
interest (spurious hits)
https://huttenhower.sph.harvard.edu/shortbred
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ShortBRED Identify
Find unique markers for interesting prots
Jim
Kaminski
Prots of Reference
interest database
True Marker
Cluster into
families
Junction Marker
https://huttenhower.sph.harvard.edu/shortbred
Identify short,
common regions
Quasi Marker
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ShortBRED Quantify
Use markers for highly specific profiling
Jim
Kaminski
Metagenome
reads ShortBRED
markers
Translated search for
high ID hits
https://huttenhower.sph.harvard.edu/shortbred
Normalize
relative
abundances
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ShortBRED: ABR in human gut metagenomes
https://huttenhower.sph.harvard.edu/shortbred
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ShortBRED: Functional profiling of
microbial genomes
http://huttenhower.sph.harvard.edu/graphlan
Thanks!
http://huttenhower.sph.harvard.edu
Human Microbiome Project 2
Alex
Ayshwarya
Xochitl
Kostic Subramanian Morgan
Casey
DuLong
Daniela
Boernigen
Lauren
McIver
Ramnik Xavier
Lita Procter
Bruce Birren
Jon Braun
Chad Nusbaum
Dermot McGovern
Clary Clish
Subra Kugathasan
Joe Petrosino
Ted Denson
Thad Stappenbeck
Janet Jansson
Human Microbiome Project
George
Weingart
Emma
Schwager
Eric
Franzosa
Boyu
Ren
Tiffany
Hsu
Ali
Rahnavard
Joseph
Moon
Jim
Kaminski
Tommi
Vatanen
Koji
Yasuda
Siyuan
Ma
Galeb
Abu-Ali
Bahar
Sayoldin
Randall
Schwager
Melanie
Schirmer
Himel
Mallick
Moran
Yassour
Alexandra
Sirota-Madi
Hera Vlamakis
Dirk Gevers
Jane Peterson
Sarah Highlander
Barbara Methe
Nicola Segata
Clary Clish
Justin Scott
Karen Nelson
George Weinstock
Owen White
Levi
Waldron
Wendy Garrett