early life dynamics of the human gut virome and …...supplementary information early life dynamics...

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.


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).


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.


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 5_6085


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. 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.


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. 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.


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


Roseburia

Chlamydiamicrovirus

Bifidobacterium

UC_Clostridiales

Akkermansia

Dorea

UC_Ruminococcaceae

Blautia

UC_Lachnospiraceae

Bacteroides

Prevotella

Roseburia

UC_Caudovirales

Dorea


UC_Clostridiales

UC_Clostridiaceae Bifidobacterium

Microviridae

Siphoviridae Podoviridae

Unclassified Caudovirales Inoviridae Myoviridae Unclassified phages

Bacteria

Bacteriophage

Negative correlation

Positive correlation


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.


early life dynamics of the human gut virome and …...supplementary information early life dynamics...

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