1. Functional profiling with HUMAnN2
Eric Franzosa
Jason Lloyd-Price
Functional profiling with HUMAnN2
Curtis Huttenhower
Galeb Abu-Ali
Ali Rahnavard
STAMPS 2017
Harvard T.H. Chan School of Public Health
Department of Biostatistics

2. The two big questions of microbial community profiling:
What are they doing?
Who is there?
(functional profiling)
(taxonomic profiling)
Like many great bioinformatics problems,
answering these questions begins with sequence search!

3. HUMAnN2 for taxon-specific metagenome and metatranscriptome functional profiling
The relative abundance of gene i in a metagenome is the number of reads j that map to a gene sequence in the family, weighted by the inverse p-value of each mapping and normalized by the average length of all gene sequences in the orthologous family.
Eric Franzosa
Lauren
McIver

4. 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
~HUMAnN1
MetaCyc pathway
Pathway abundance & coverage
PWY-7221: GTP biosynthesis
200.35 1
PWY-7221: GTP biosynthesis|Bacteroides_caccae
120.23
PWY-7221: GTP biosynthesis|Bacteroides_dorei
11.12

5. 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 >2K human metagenomes (HMP1-II, 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

6. And it works on non-human meta’omes, too
Luke Thompson

7. Quantifying the diversity of species contributing a function within and across subjects
low
between-subject diversity
high
low
simple,
consistent
simple,
variable
within-subject diversity
A pathway’s contributional alpha-diversity is calculated from the distribution of taxa providing it (DNA or RNA) within a community; contributional beta-diversity is the corresponding comparison between communities.
complex,
consistent
complex,
variable
high

8. HUMAnN2 reveals unusual “relative expression” in paired metatranscriptomes & metagenomes
Sucrose degradation follows a complex attribution pattern across ~200 human gut metagenomes…
In collaboration with
the STARR Consortium
& HPFS cohort
…but its expression can be dominated by a single species in paired gut metatranscriptomes!

9. The “HMP2” IBD Multi’omics Data resource
With Ramnik Xavier

10. The IBD Multi’omics DataBase
Cesar Arze

11. The IBD metatranscriptome in the HMP2 IBDMDB
117 Subjects:
59 Crohn’s Disease
34 Ulcerative Colitis
24 non-IBD Controls
Gender:
57 Female
59 Male
1 unknown
Cohorts:
32 MGH adult new onset
30 Cedars-Sinai adult establ.
31 Cincinnati peds new onset
11 Emory peds new onset
13 MGH peds new onset
Melanie Schirmer

12. Different microbes can transcribe shared pathways
HISDEG-PWY: L-histidine degradation I
Histidine is an α-amino acid that is used in the biosynthesis of proteins
A. putredinis has been implicated in IBD
Major contributor to transcription in subsets of IBD patients

13. PWY-7094: fatty acid salvage
Pathways can be contributed by different microbes over time
PWY-7094: fatty acid salvage
Faecalibacterium prausnitzii
Time-courses for individual patients:
CD Patient 1
CD Patient 2

14. https://bitbucket.org/biobakery/biobakery/wiki/humann2
HUMAnN2 tutorial

16. HUMAnN2 synthetic evaluation (genes)
…and is ~3x faster
~2.1 hours
~0.7 hours
(10M reads,
8 cores)
HUMAnN2 tiered search is more accurate…
Comprehensive search suffers from spurious hits
...and
provides
accurate
per-species
quantification!
Compare exp. vs. obs. gene abundance 1x
Staggered abundance
~0.1x to 100x coverage
Synthetic human gut metagenome (top 20 species)

17. HUMAnN2 real-world performance

18. Considerations for paired metatranscriptomes & metagenomes
$ humann2_rna_dna_norm --input_dna <DNA genefamilies file> input_rna <RNA genefamilies file> --output_basename <basename of the 3 output files>
Calculates RNA/DNA abundance ratios
Smooths the RNA and DNA abundances prior to taking the ratio
Also outputs smoothed RNA and DNA files
UniRef90_R6K3Z5: IMP dehydrogenase
2.02
UniRef90_R6K3Z5: IMP dehydrogenase|Bacteroides_caccae
5.96
UniRef90_R6K3Z5: IMP dehydrogenase|Bacteroides_dorei
3.82
UniRef90_R6K3Z5: IMP dehydrogenase|Bacteroides_ovatus
1.80
UniRef90_R6K3Z5: IMP dehydrogenase|Bacteroides_stercoris
0.87
UniRef90_R6K3Z5: IMP dehydrogenase|Bacteroides_vulgatus
0.34
UniRef90_R6K3Z5: IMP dehydrogenase|unclassified
1.96