Presented by Roxana Hickey at the Gordon Research Conference on Microbial Population Biology, July 2013

1. Roxana Hickey,1,2 Sam Hunter,2 Matthew Settles,2 Bing Ma,3 Garry Myers,3 Jacques Ravel3 and Larry Forney1,2
1Department of Biological Sciences and 2Institute for Bioinformatics and Evolutionary Studies (IBEST), University of Idaho, Moscow, Idaho, USA
3Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, Maryland, USA
why a microarray for microbiome analysis?
design & production of the V-chip
• 200 vaginal bacterial
species (336 strains)
• genome sequences in
public databases
• 812,653 genes/ORFs
• 693,707 unique
• <10 bp or >9,999 bp
excluded (n=240)
Raw data input
• Cd-hit clustering: 80%
identity, 80% coverage
• 473,709 gene/ORF clusters
• Representative sequences
from multiple sequence
alignment in MUSCLE
Sequence
clustering &
alignment
• 3-plex 4.2M probe array
• 1.2M per subarray
• 2-5 60-mer probes per
cluster
• 307,860/473,709 clusters
represented
• 716 human immunity genes
(3,580 probes)
Probe design
mock communities: bacterial + human genomic DNA
Table 1. Composition of mock communities tested on the V-chip microarray
Mock community! A! B! C! D! E! F! G! H! I!
Species! Proportion of genomic DNA in mixture!
Anaerococcus hydrogenalis (vaginal isolate)
0.200
0.100
0.010
0.010
0.001
-
-
-
-
Anaerococcus tetradius (vaginal isolate)
0.200
0.100
0.010
0.010
0.001
-
-
-
-
Atopobium vaginae ATCC BAA-55
0.200
0.100
0.010
0.010
0.001
-
-
-
-
Finegoldia magna (vaginal isolate)
0.200
0.100
0.010
0.010
0.001
-
-
-
-
Gardnerella vaginalis ATCC 14018
0.200
0.100
0.010
0.010
0.001
-
-
-
-
Lactobacillus crispatus ATCC 33820
-
0.500
0.950
0.050
0.050
0.050
0.010
1.000
-
Homo sapiens female genomic DNA
-
-
-
0.900
0.945
0.950
0.990
-
1.000
vaginal swabs: V-chip vs. 16S rRNA pyrosequencing
Subject 1 (L. iners)
Subject 2 (L. crispatus)
Figure 2. Daily temporal
dynamics of vaginal bacterial
communities in two women
over 10 weeks, along with
associated subject metadata.
Subject 1’s microbiota
remained relatively stable
throughout the study, while
Subject 2 experienced a
drastic alteration of community
composition following vaginal
intercourse in week 8.
1 2A
2B
A B
Figure 3. Relative abundances of species
detected on the V-chip compared to 16S
rRNA V1-V2 pyrosequencing.
Table 2. Pearson correlation coefficients
for V-chip vs. 16S rRNA species relative
abundance
! V-chip vs. 16S rRNA
1
!
DNA vs. 16S! cDNA vs. 16S!
Sample 1
1.00
0.92
Sample 2A
0.53
0.59
Sample 2B
0.84
0.69
Overall
0.92
0.84
1 Hypervariable V1-V2 region of 16S rRNA genes were
determined previously by pyrosequencing
Table 3. Pearson correlation coefficients for V-chip vs. in
silico mapping of Illumina RNA-Seq reads against probes
Genus (upper
diag.) / Species
(lower)!
Sample 1!
cDNA!
Sample 1!
reads!
Sample 2A
cDNA!
Sample 2A
reads!
Sample 1 cDNA
1.00
0.71
0.37
0.31
Sample 1 reads
0.75
1.00
0.33
0.53
Sample 2A cDNA
0.36
0.31
1.00
0.79
Sample 2A reads
0.20
0.25
0.77
1.00
Figure 4. Number of species-specific gene clusters with a
greater than two-fold change between time points in the
metatranscriptome of samples 2A and 2B.
in silico hybridization: V-chip vs. Illumina RNA-Seq
using V-chip to measure transcriptional response
Ø High-throughput DNA sequencing technologies have drastically reduced costs
and improved efficiency, but bioinformatics expertise and computational time
remain a barrier to rapid analysis and accessibility
Ø Microarrays are a faster, more straightforward alternative to sequencing that
can more readily be utilized in smaller research studies and clinical settings
Ø V-chip microarray developed for explorative analysis of the vaginal microbiome
vaginal swabs: metagenomes & metatranscriptomes
conclusions
Ø V-chip shows high-level agreement with sequencing-based techniques
Ø Utility as a discovery tool with a variety of potential applications:
• Identify genes or taxa that contribute to community ecological function or
health outcomes of the host
• Screen samples for interesting patterns to select manageable subset for in
depth investigation
acknowledgments
RH is supported by a fellowship from the University of Idaho Bioinformatics and Computational Biology
Graduate Program and grant U19 AI084044 from the National Institutes of Health. The research study was
supported by grants UH2 AI083264 and U01 AI070921 from the National Institute of Allergy and Infectious
Diseases, National Institutes of Health. The authors are grateful to Daniel New (IBEST Genomics Core),
Renee Nuhn (Forney lab) and Li Fu (Ravel lab) for their technical assistance.
Figure 1. Relative abundances of species
in mock community samples detected by
species-specific probes on the V-chip (left)
compared to expected (right). Relative
abundance was determined from RMA-
normalized hybridization signal intensity
values.
Mock communities of genomic DNA (bacterial + human) tested to evaluate
qualitative and quantitative capabilities of the V-chip
Metagenome (DNA) & metatranscriptome (mRNA è cDNA) of three vaginal
swabs tested to evaluate utility of V-chip for vaginal microbial communities
High correlation of species relative abundance between cDNA hybridization
on V-chip and in silico mapping of RNA-Seq reads
Ø Illumina RNA-Seq
reads (100 bp)
mapped to V-chip
probes in silico using
Bowtie (v0.12.9)
Ø ~2-3% of total reads
mapped to V-chip
probes
V-chip sensitive and specific, but not
quantitative, for detecting species-
specific genes in mock communities
V-chip useful for
performing qualitative
comparisons of samples
As with mock communities, V-chip sensitive but not highly quantitative for
detecting species-specific genes in vaginal microbial communities
Ø V-chip sensitive to low-abundance
bacteria, but more useful as
qualitative rather than quantitative
tool, particularly for highly skewed
communities
Ø Comparison of relative abundance of
42 bacterial species in metagenome
and metatranscriptome on V-chip
with previous 16S rRNA V1-V3
pyrosequencing results
Ø Many species-specific
genes differentially
expressed, reflecting
changes in community
composition over time
interval