early life dynamics of the human gut virome and bacterial...
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Supplementary Information
Early life dynamics of the human gut virome and bacterial microbiome in infants
Efrem S. Lim1,2, Yanjiao Zhou3,4, Guoyan Zhao1, Irma K. Bauer3, Lindsay Droit1,2, I. Malick Ndao3, Barbara B. Warner3, Phillip I. Tarr1,3, David Wang1,2, Lori R. Holtz3
1Department of Molecular Microbiology, Washington University School of Medicine, St Louis, Missouri
2 Department of Pathology & Immunology, Washington University School of Medicine, St Louis, Missouri
3Department of Pediatrics, Washington University School of Medicine, St Louis, Missouri
4Current address: The Jackson Laboratory for Genomic Medicine, Farmington, Connecticut; Channing Division of Network Medicine, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts
Address correspondence to: L.R.H. ([email protected]), D.W. ([email protected])
Nature Medicine: doi:10.1038/nm.3950
Supplementary Figure 1a
Fever/vomiting/diarrheaAntibiotic use
Stool samplingSolid food
0 200 400 600 800Age (days)
A1
A2
B1
B2
C1
C2
D1
D2
0 3 6 12 18 24105
106
107
Timepoint (month)0 3 6 12 18 24
105
106
107
Timepoint (month)
MDAAverage reads per sample: 549,301
(± 207,521 s.d.)
SIAAverage reads per sample: 551,592
(± 229,210 s.d.)
No.
of r
eads
b
cesarean section breastfed only
breastfed and formula
formula only
breastfed and formula
Zygosity Delivery mode Diet
♂
♀
♂
♀
♂
♂
♂
♂
MaleFemale♀
♂
dizygotic
dizygotic
dizygotic
monozygotic
vaginal
cesarean section
cesarean section
Day 404
Day 67
N/A
Day 43
Weaned
Supplementary Figure 1. Infant cohort description and sequencing depth. (a) Timeline represents stools collected from 8 healthy (4 twin pairs) infants, as indicated by closed circles. Grey diamonds indicate time when solid food was introduced. Reported cases of fever, vomiting or diarrhea are indicated with grey inverted triangles. Reported antibiotic use is indicated with a white triangle. (b) Number of sequencing reads obtained for each sample for the MDA method (left) and SIA method (right). SIA, sequence independent DNA and RNA amplification; MDA, multiple displacement amplification; s.d., standard deviation.
Nature Medicine: doi:10.1038/nm.3950
PC3 (7.55%)0.2- 0.3 - 0.2 - 0.1 0 0.1
- 0.2
- 0.1
0
0.1
0.2
PC
2 (9
.02%
)
PC3 (7.55%)0.2- 0.3 - 0.2 - 0.1 0 0.1
- 0.2
- 0.1
0
0.1
0.2
ABCD
Twins
PC
2 (9
.02%
)
0.2
-0.2
-0.1
0
0.1
-0.6PC1 (39.25%)
-0.4 -0.2 0 0.40.2
0.2
-0.2
-0.1
0
0.1
-0.6PC1 (39.25%)
-0.4 -0.2 0 0.40.2
036121824
Age (months)
Supplementary Figure 2a
b
PC
2 (9
.02%
)
PC
2 (9
.02%
)
Supplementary Figure 2. Principal coordinate analysis (PCoA) of viromes. PCoA analysis of unweighted bray-curtis distance of virome communities (eukaryotic viruses and bacteriophages; genera) (n = 48 sampling time points). The variance explained by each PC is indicated on the axis. Specimens from the same twin pairs are indicated in (a), and age from 0 – 24 months is shown in the color gradient in (b).
Nature Medicine: doi:10.1038/nm.3950
a
b
B1-6B2-6C1-3C2-3A1-6A2-6
VP0 VP3 VP1 2A 3A3B
3C5’ UTR 3’ UTR
AAAAA2B 2C 3D
SequencingRT-PCRParechovirus
Enterovirus
0.06
C2-18
Human coxsackievirus A7 (GU942820)
Human coxsackievirus A16 (JQ746660)
B2-18
Enterovirus A (NC_001612)
C1-18
Enterovirus B (KF878966)
Rhinovirus B (DQ473485)
B1-18
C2-3
Human enterovirus B (NC_001472)
92
100
93
99100
100
Shared
Shared
B1-18B2-18C1-18
C2-3C2-18
VP3 VP1 2A3A 3B
3C 3D5’ UTR 3’ UTR
AAAAA2B 2CVP2VP4
0 3 6 12 18 240
5
10
15
A1 AlphatorquevirusBetatorquevirusBetatorquevirusGammatorquevirusUnclassified anellovirus
0 3 6 12 18 240
5
10
15
A2
0 3 6 12 18 2402468
10
B1
0 3 6 12 18 2402468
10
B2
0 3 6 12 18 240
1020304050
C1
0 3 6 12 18 240
1020304050
C2
0 3 6 12 18 240
5
10
15
D1
0 3 6 12 18 240
5
10
15
D2
No.
of a
nello
viru
ses
Age (months)
e
f PCR (+) PCR (-)
Sequencing (+)
Sequencing (-)
7
1351
1
0 25 50 75 1000
100
200
300
% identity
No.
of p
airw
ise
com
paris
ons
d
y = -3.41x + 39.35R2 = 0.998
Copy number
Thre
shol
d cy
cle
(Ct)
SampleqRT-PCR
Ct
qRT-PCRcopy no./15mgfecal specimen
B1-6B2-6C1-3C2-3A1-6A2-6
D2-12D1-12
25.6925.4324.1722.7527.9729.5929.7035.09
10,11012,05828,19173,8262,17873067518
c Parechovirus qRT-PCR standard curve
100 101 102 103 104 105 106 1070
10
20
30
40
Sequencingreads
2105,299307
1,24531000
Supplementary Figure 3
Supplementary Figure 3. Characterization of eukaryotic RNA and DNA viruses. (a) Contigs mapped to the parechovirus genome are shown. Contigs assembled from sequencing reads are indicated in white, and sequence regions obtained from RT-PCR fragments are indicated in grey. (b) Contigs mapped to enterovirus genome are shown. Phylogenetic analysis of enterovirus amino acid sequences from concat-enated 2BC and 3CD regions. Maximum likelihood analyses was performed, bootstrap support are indicated above branches. (c) Standard curve of the qRT-PCR assay for human parechovirus was performed in triplicate. Error bars indicate the standard deviation. Best-fit line, equation and R-squared values are shown. Comparison of the number of parechovirus sequencing reads to qRT-PCR assay threshold cycle (Ct) for parechovirus-positive samples and the viral load measurement (copy number/15mg of fecal specimen) are shown on the right. (d) Histogram plot of the pairwise % identity comparisons of the anellovirus genome contigs. (e) Distribution of anelloviruses from each infant is shown. The genera distribution is colored as indicated. (f) Concordance between PCR assay and in silico sequence mapping was assessed using a 2x2 contingency table. The PCR assay was based on screening results of an alphatorquevirus, betatorquevirus and gammatorquevirus.
Nature Medicine: doi:10.1038/nm.3950
MDA SIA
0
0.5
1.0
Mic
rovi
ridae
abu
ndan
ce
b
0.2
Contig 55_5491
Contig 105_6165
Chlamydia phage Chp2 (NC_002194)
Bdellovibrio phage phiMH2k (NC_002643)
Contig 3_4870
Contig 2_6739
Chlamydia phage 4 (AY769964)
Chlamydia phage 3 (AJ550635)
Contig 19_5732Contig 17_5979
Marine gokushovirus SOG1 (KC131024)
Contig 15_5695
Contig 4_4863
Microvirus CA82 (NC_015785)
Chlamydia phage PhiCPAR39 (AE002163)
PhiX174 (NC_001422)
Marine gokushovirus SI2 (KC131023)
Chlamydia phage PhiCPG1 (NC_001998)
Chlamydia phage Chp1 (BCP1)
Contig 18_5750Contig 3_7260
Contig 5_6085
Contig 11_3897Contig 16_4946
Contig 1_6081
d
a
0 3 6 12 18 24
0
5
10
15
Age (months)
Mic
rovi
ridae
rich
ness
c*
**
0 3 6 12 18 24 0 3 6 12 18 24 0 3 6 12 18 24 0 3 6 12 18 24 0 3 6 12 18 24 0 3 6 12 18 24 0 3 6 12 18 24 0 3 6 12 18 240
0.2
0.4
0.6
0.8
1.0
A1 A2 B1 B2 C1 C2 D1 D2
Age (months)
Rel
ativ
e ab
unda
nce
Unclassified SiphoviridaeStreptococcus phage 5093Croceibacter phage P2559SBurkholderia phage KS9Streptococcus phage P9Enterobacteria phage lambdaStreptococcus phage SMPBacillus phage BCJA1cRiemerella phage RAP44Rhodococcus phage ReqiPoco6Streptococcus phage PH15Streptococcus phage YMC-2011LambdalikevirusSiphoviridaeClostridium phage PhiS63
Siphoviridae
Unidentified phageEnvironmental Halophage eHP-12Streptococcus phage 2167Unclassified phages
Unclassified bacteriophages
Cellulophaga phage phi4:1Enterobacteria phage vBCellulophaga phage phi18:3Unclassified Sp6likevirusBacillus phage MG-B1
Podoviridae
unclassified MyoviridaeCellulophaga phage phiSMBacillus phage BCD7Bacillus phage GClostridium phage phiC2MyoviridaeRhizobium phage RR1-B
Myoviridae
Propionibacterium phage B5Enterobacteria phage f1
Inoviridae
MicroviridaeGokushovirinaeMicroviridae phi-CA82ChlamydiamicrovirusMarine gokushovirusUncultured MicroviridaeBdellovibrio phage phiMH2K
Microviridae
CaudoviralesCaudovirales
Supplementary Figure 4
Supplementary Figure 4. Analysis of the bacteriophage community.(a) Relative abundance of bacteriophages genera. Only bacteriophage taxa with greater than 0.05 relative abundance is shown. (b) Comparison of Microviridae abundance of the MDA and SIA methods from specimens at 24 months age is shown (n = 8 infants). The abundance observed for each sample is indicated with a line connecting the paired method. (c) Richness of Microviridae bacteriophage species is shown (n = 8 infants). Statistical significance was assessed by Wilcoxon test (paired, non-parametric); * P = 0.01 - 0.05, ** P <0.01. (d) Maximum likelihood phylogenetic analyses of the Microviridae bacteriophages identified in this study are shown. Phylogenetic relationships were inferred from the amino acid sequence alignment of the major capsid protein.
Nature Medicine: doi:10.1038/nm.3950
a
No. of samples
Bact
eria
l ric
hnes
s
0 mo3 mo6 mo12 mo18 mo24 mo
0 2 4 6 80
500
1000
1500
2000
036121824
Age (months)
PC3 (5.72%)
PC
2 (9
.47%
)
0.5
0.4
0.3
0.2
0.1
0
- 0.1
- 0.2
- 0.3- 0.3 - 0.2 - 0.1 0 0.1 0.2 0.3 0.4
PC1 (15.98%)
PC
2 (9
.47%
)
0.5
0.4
0.3
0.2
0.1
0
- 0.1
- 0.2
- 0.3- 0.4 - 0.3 - 0.2 - 0.1 0 0.1 0.2 0.3 0.4
ABCD
Twins
PC3 (5.72%)
PC
2 (9
.47%
)
0.5
0.4
0.3
0.2
0.1
0
- 0.1
- 0.2
- 0.3- 0.3 - 0.2 - 0.1 0 0.1 0.2 0.3 0.4
c
b
0 3 6 12 18 24 0 3 6 12 18 24 0 3 6 12 18 24 0 3 6 12 18 24 0 3 6 12 18 24 0 3 6 12 18 24 0 3 6 12 18 24 0 3 6 12 18 240
0.2
0.4
0.6
0.8
1.0
Rel
ativ
eab
unda
nce
ClostridiaUnclass Clostridiales
Finegoldia
ClostridiumUnclass Clostridiaceae
Unclass LachnospiraceaeBlautiaRuminococcusDoreaRoseburiaCoprococcusLachnospiraEpulopiscium
Tissierellaceae
Clostridiaceae
Lachnospiraceae
Clostridium
Unclass RuminococcaceaeRuminococcusOscillospiraFaecalibacterium
VeillonellaMegamonasMegasphaeraUnclass DialisterUnclass Veillonellaceae
ClostridiaPeptostreptococcaceae
Ruminococcaceae
Veillonellaceae
Unclass ErysipelotrichaceaeEubacterium
Bifidobacterium
Corynebacterium
Paraprevotella
Bacteroides
Parabacteroides
Prevotella
ErysipelotrichiErysipelotrichaceae
ActinobacteriaBifidobacteriaceae
Corynebacteriaceae
BacteroidiaParaprevotellaceae
Bacteroidaceae
Porphyromonadaceae
Prevotellaceae
Unclass Planococcaceae
Staphylococcus
StreptococcusLactococcus
Unclass Enterobacteriaceae
Acinetobacter
Haemophilus
Pseudomonas
Akkermansia
Fusobacterium
BacilliPlanococcaceae
Staphylococcaceae
Streptococcaceae
GammaproteobacteriaEnterobacteriaceae
Moraxellaceae
Pasteurellaceae
Pseudomonadaceae
VerrucomicrobiaeVerrucomicrobiaceae
FusobacteriiaFusobacteriaceae
D2
Age (months)
D1C2C1B2B1A2A1
PC1 (15.98%)
PC
2 (9
.47%
)
- 0.4 - 0.3 - 0.2 - 0.1 0 0.1 0.2 0.3 0.4- 0.3
0.5
0.4
0.3
0.2
0.1
0
- 0.1
- 0.2
d
Supplementary Figure 5
Supplementary Figure 5. Analysis of the bacterial community.(a) PCoA plots of bacterial unifrac distance matrices (n = 48 sampling time points). The variance explained by each PC is indicated on the axis. Color gradient represents community progression with age. (b) PCoA plots of unifrac distance matrices (n = 48 sampling time points). Twin pairs are colored as indicated. (c) Rarefaction curves of bacterial richness (OTUs) versus an increasing number of specimen subsamplings with replacement are shown. The curves represent the average of 500 iterations at each depth of samples. (d) Relative abundance of the 40 most abundant bacterial genera is shown.
Nature Medicine: doi:10.1038/nm.3950
VeillonellaBacteroides
UC_phages
UC_Clostridiaceae
Staphylococcus
Streptococcus
UC_Ruminococcaceae
UC_Enterobacteriaceae
AkkermansiaPhiCD119likevirus
UC_Erysipelotrichaceae
Dorea
UC_Clostridiales
UC_LachnospiraceaeClostridium
Ruminococcus
Bacteroides
Blautia
UC_Myoviridae
RoseburiaPrevotella
Roseburia
VeillonellaUC_Clostridiaceae
Clostridium
RuminococcusUC_ErysipelotrichaceaeUC_Clostridiales
Blautia
Roseburia DoreaBifidobacterium
UC_ErysipelotrichaceaePrevotella
Ruminococcus
AkkermansiaBacteroides
Staphylococcus
UC_Lachnospiraceae
UC_Siphoviridae
UC_Lambda-like_viruses
LambdalikevirusUC_Clostridiaceae
UC_Clostridiaceae
DoreaUC_Inoviridae
Inovirus
Ruminococcus
Bifidobacterium
UC_Ruminococcaceae
UC_ErysipelotrichaceaeClostridium
UC_Clostridiales
Prevotella
Clostridium Roseburia
Ruminococcus
Blautia
Veillonella
UC_Lachnospiraceae
BacteroidesVeillonellaAkkermansia
UC_Clostridiales
Bacteroides
UC_Podoviridae
Bifidobacterium
UC_Lachnospiraceae
Prevotella
Dorea
Blautia
UC_Ruminococcaceae
Akkermansia
UC_Ruminococcaceae
Clostridium
Veillonella
Streptococcus
Streptococcus
StaphylococcusUC_Enterobacteriaceae
Spiromicrovirus
UC_Microviridae
Bdellomicrovirus
Veillonella
Prevotella
Bacteroides
Clostridium
UC_Gokushovirinae
UC_Clostridiaceae
UC_Lachnospiraceae
Microvirus
Akkermansia
Blautia
Ruminococcus
UC_Ruminococcaceae
Bifidobacterium
UC_Erysipelotrichaceae
Roseburia
Chlamydiamicrovirus
Bifidobacterium
UC_Clostridiales
Akkermansia
Dorea
UC_Ruminococcaceae
Blautia
UC_Lachnospiraceae
Bacteroides
Prevotella
Roseburia
UC_Caudovirales
Dorea
UC_Erysipelotrichaceae
UC_Clostridiales
UC_Clostridiaceae Bifidobacterium
Microviridae
Siphoviridae Podoviridae
Unclassified Caudovirales Inoviridae Myoviridae Unclassified phages
Bacteria
Bacteriophage
Negative correlation
Positive correlation
Supplementary Figure 6
Supplementary Figure 6. Bacteriophage-bacteria network analysis. Network between bacteriophage genera (red node) and bacteria genera (grey node) is plotted by indicated bacteriophage family. Linear mixed model was applied to identify significant changes in relative abundance over time. Negative correlations are shown with blue lines and positive correlations are shown in orange lines. UC, unclassified.
Nature Medicine: doi:10.1038/nm.3950