Contents

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ES 1A: Samples and Libraries

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All reads were preprocessed for quality, 5'-bias and poly-A tails. Preprocessed HiSeq libraries for HuH7 and R06E-J samples were pooled for assembly.

Additionally, all nine R06E-J samples were pooled and sequenced on a single MiSeq lane with a length of 300 bp and in paired-end mode. Read coverage was calculated on an expected eukaryotic transcriptome size of 75 Mbp.

SampleRead NumbersCoverageFastQC read statistics
rawtrimmedrawtrimmedrawtrimmed
HuH7: HiSeq Samples
HuH7-MOCK-3h 40,529,77038,408,644 54X51X forward,reverseforward,reverse
HuH7-MOCK-7h 38,008,32035,958,522 51X48X forward,reverseforward,reverse
HuH7-MOCK-23h 39,006,44236,866,384 52X49X forward,reverseforward,reverse
HuH7-EBOV-3h 34,429,92232,777,274 46X44X forward,reverseforward,reverse
HuH7-EBOV-7h 49,982,53646,619,978 67X62X forward,reverseforward,reverse
HuH7-EBOV-23h 53,034,36050,112,880 71X67X forward,reverseforward,reverse
HuH7-MARV-3h 44,197,62241,778,332 59X56X forward,reverseforward,reverse
HuH7-MARV-7h 48,264,54445,678,616 64X61X forward,reverseforward,reverse
HuH7-MARV-23h 36,284,71434,261,494 48X46X forward,reverseforward,reverse
HuH7-Pooled 382,332,302362,462,124 510X483X forward,reverseforward,reverse
R06E-J: HiSeq Samples
R06E-J-MOCK-3h 50,396,27647,815,138 67X64X forward,reverseforward,reverse
R06E-J-MOCK-7h 44,016,80041,351,362 59X55X forward,reverseforward,reverse
R06E-J-MOCK-23h 48,464,38245,474,028 65X61X forward,reverseforward,reverse
R06E-J-EBOV-3h 41,450,60639,376,162 55X53X forward,reverseforward,reverse
R06E-J-EBOV-7h 38,362,12636,005,676 51X48X forward,reverseforward,reverse
R06E-J-EBOV-23h 39,292,72237,350,338 52X50X forward,reverseforward,reverse
R06E-J-MARV-3h 37,490,48635,538,058 50X47X forward,reverseforward,reverse
R06E-J-MARV-7h 48,645,54445,862,636 65X61X forward,reverseforward,reverse
R06E-J-MARV-23h 45,569,35643,291,468 61X58X forward,reverseforward,reverse
R06E-J-Pooled 393,021,564372,082,040 524X496X forward,reverseforward,reverse
R06E-J: MiSeq Sample
R06E-J-MiSeq 38,163,20038,028,488 153X152X forward,reverseforward,reverse



ES 1B: De novo transcriptome assembly of Rousettus aegyptiacus

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AssemblyContigsContigs >1000 bpmax. Contig (bp)ParameterDownload (*.tar.gz)
Velvet/Oases* 370,200 180,458 36,073 k = 45,55,65,75; multiple kmer-assemblies of Velvet merged by Oases like described in manual link (95 Mb)
SoapDenovo-Trans* 699,418 147,144 34,307 k = 25,35,45,55,65,75; multiple kmer-assemblies merged by cd-hit-est (-c 0.95) link (189 Mb)
Trinity* 484,826 188,534 30,056 k = 25 (fix) link (172 Mb)
ABySS/TransABySS* 790,204 169,324 21,424 k = 25,35,45,55,65,75; multiple kmer-assemblies of ABySS merged by TransABySS pipeline link (192 Mb)
Mira** 162,861 21,987 14,119 overlap graph approach; job = est,denovo,accurate link (33 Mb)
Clustered (cd-hit-est) 977,787 277,595 36,073 -c 0.95 link (290 Mb)

* 9 different samples were sequenced individually on a Illumina HiSeq with 100bp, paired end; after sequencing pooled for assembly

** all 9 samples were pooled and normalized before library preparation, then sequenced on a Illumina MiSeq, 300bp, paired end



QUAST: full report statistics




ES 2A: Hierarchical clustering of log2 gene-expression fold-changes upon infection of human cells with EBOV or MARV

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Hierarchical clustering of log2 gene-expression fold-changes upon infection with EBOV or MARV, respectively. Deregulated genes (padj < 0.1) with an absolute log2 expression fold-change greater than five in at least one of the contrasts (infection versus Mock control at a specific time point post infection, p.i.) have been considered for hierarchical clustering of log2 gene expression fold changes.

click for heatmap


ES 2B: Differential gene expression: Overview

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Pairwise Comparisons

Overview of different comparisons within human (bat) samples and between the two species. Click on button to see all comparisons and links to the corresponding tables with all differential expressed genes with a padj < 0.1. Same colors indicate that the results are listed in the same table.



MOCK-3h MOCK-7h MOCK-23h EBOV-3h EBOV-7h EBOV-23h MARV-3h MARV-7h MARV-23h
MOCK-3h

MOCK-7h

MOCK-23h

EBOV-3h

EBOV-7h

EBOV-23h

MARV-3h

MARV-7h

MARV-23h



Comparisons including Replicates

Overview of different comparisons between Mock and filovirus (EBOV, MARV) samples using replicates. Click on button to see all comparisons and links to the corresponding tables with all differential expressed genes filtered by a padj <= 0.05. Same colors indicate that the results are listed in the same table.



MOCK FILO-3h FILO-7h FILO-23h
MOCK

FILO-3h

FILO-7h

FILO-23h

ES 2C: Comparisons between time points 3h, 7h and 23h of Mock, EBOV and MARV samples within human and bat

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Differential gene expression of HuH7/R06E-J MOCK, EBOV and MARV samples compared at 3h, 7h and 23h, respectively. These tables show the differential expression within MOCK, EBOV and MARV and between the different time points, separate for human and bat. Tables are sorted by fold change.




ES 2D: Differential gene expression: Comparisons between Mock, EBOV and MARV samples

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All tables are sorted by fold change.

Comparison Table link Download table Description
HuH7 Mock/EBOV
@3h,7h,23h
Go click Differential gene expression of HuH7 MOCK and EBOV samples compared at 3h, 7h and 23h post infection.
HuH7 Mock/MARV
@3h,7h,23h
Go click Differential gene expression of HuH7 MOCK and MARV samples compared at 3h, 7h and 23h post infection.
HuH7 EBOV/MARV
@3h,7h,23h
Go click Differential gene expression of HuH7 EBOV and MARV samples compared at 3h, 7h and 23h post infection.
R06E-J Mock/EBOV
@3h,7h,23h
Go click Differential gene expression of R06E-J MOCK and EBOV samples compared at 3h, 7h and 23h post infection.
R06E-J Mock/MARV
@3h,7h,23h
Go click Differential gene expression of R06E-J MOCK and MARV samples compared at 3h, 7h and 23h post infection.
R06E-J EBOV/MARV
@3h,7h,23h
Go click Differential gene expression of R06E-J EBOV and MARV samples compared at 3h, 7h and 23h post infection.



ES 2E: Comparisons between human and bat

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We compared differential expression between human and bat EBOV and MARV infected samples at 3h, 7h and 23h poi. Additionally, we used EBOV and MARV samples at same time points as replicates with DESeq to compare the characteristics of filovirus infection between human and bat.




ES 2F: Human MOCK samples compared to EBOV+MARV cells using replicates

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Differential gene expression of HuH7 MOCK samples (3h, 7h and 23h as replicates) compared to EBOV+MARV samples (as replicates, called FILO) at 23h. For 3h and 7h no differential expressed genes were found with a padj < 0.05.




ES 2G: Human vs Bat samples including replicates

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Differential gene expression of HuH7 MOCK/EBOV/MARV samples compared to R06E-J MOCK/EBOV/MARV samples at 3h, 7h and 23h (filtered by a padj < 0.05). Within this experiment we compared:

  1. HuH7 MOCK3h,7h,23h vs R06E-J MOCK3h,7h,23h
  2. HuH7 FILOEBOV-3h,MARV-3h vs R06E-J FILOEBOV-3h,MARV-3h
  3. HuH7 FILOEBOV-7h,MARV-3h vs R06E-J FILOEBOV-7h,MARV-3h
  4. HuH7 FILOEBOV-23h,MARV-3h vs R06E-J FILOEBOV-23h,MARV-3h
and additionally filterd results of (2), (3) and (4) by removing genes which were also found as differential expressed between MOCK samples (1):




ES 2H: Comparison of de novo predicted gene loci of human and bat

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We used the first step of the Cufflinks pipeline to detect possible gene loci de novo based on Tophat mapped read data. We further compared the differential expression on each gene loci between different time points and within one condition as well as at same time points and different conditions. All results for human and bat (norm. read counts, log2 fold changes, padj) are listed in the following tables, respectively.




ES 2I: Comparison of ncRNA genes within human and bat

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We compared the differential expression of each ncRNA between different time points and within one condition as well as at same time points and different conditions. All results for human and bat (norm. read counts, log2 fold changes, padj) are listed in the following tables, respectively.




ES 4: Common genes during filovirus infection

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ES 4A: Summary download (pdf)




ES 4B: Scatterplots

To compare the differential expression of Mock/EBOV and Mock/MARV in human and bat cells, log2 fold changes (FC) as computed by DEseq were visualized using scatter plots in R (x-value: FC of Mock/EBOV; y-value: FC of Mock/MARV). The scatterplots were overlaid with contour plots for a two-dimensional kernel estimate (kde2d; MASS package) using the default parameters. Outliers were labeled with the respective gene names.

3h 7h 23h
Human
click for PDF click for PDF click for PDF
Bat
click for PDF click for PDF click for PDF


ES 4C: Group plots/Timeseries

To identify co-regulated genes over the course of the three timepoints (3h, 7h, 23h) the differential expression of Mock/EBOV (x-value) and Mock/MARV (y-value) was visualized for all genes using lines. The expression changes between the three time points define an angle that is subsequently used to group genes that have approximately the same or approximately inverse expression time courses. Note, that the angle between the three time points is rotation invariant.

3h 7h 23h
Human
click for PDF click for PDF click for PDF
Bat
click for PDF click for PDF click for PDF



ES 6: Pathways

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How to read pathway figures

Here, we describe how to read the gene-regulation boxes next to each gene, to fastly follow the pathways and their regulations over all time points, infections and hosts. Shown are the most significant genes according to expression level differences. To give an example how to read the boxes displayed here and in the different pathways in the electronical supplement, we use DUSP8 for explanation:

The upper half (pink) displays human samples, the lower half (blue) refers to bat samples. DUSP8 is in human Mock cells is slightly down-regulated from 3 to 7h, furthermore slightly down-regulated from 7 to 23h. The minimum of transcripts for all three timepoints in human Mock cells is read_max=31. In EBOV-infected human cells we observe from 3-7h a slight down-regulation, although the manual profile impression leads a slight up-regulation. From 7 to 23h we see a dramatic up-regulation of 25X leading to a read_max of 626. For MARV-infected human cells we see from 3-7h a slight down-regulation similar to EBOV-infected human cells from 3-7h, however this mRNA level stays from 7 to 23h constant. In bats we observe in Mock from 7 to 23h a slight down-regulation, similar to EBOV-infected bat cells from 3h to 7h. In a later stage, we observe a slight up-regulation, just as during the complete MARV-infected bat cells. The regulation level is only changing slightly as depicted in the manual search.

Furthermore, we framed each gene red, when being differentially expressed in human between the different time points. We added a green frame, when a difference between EBOV and MARV was observed in the general expression and we used blue to mark differences between human and bat transcriptome levels. The line width of the frame corresponds to the significance of the expressional change.

NA - not assigned; no bat homologous gene was detected. When the expression level changes by >15% an arrow indicates the regulation; when the expression level changes by more than 100% (2-fold transcription) a number beside the arrow indicates the fold change.





Investigated Pathways

Overview of all kingpins and related interactions
svg
pdf and description
click for PDF
Mitogen-activated protein kinase (MAPK)
svg
pdf and description
click for PDF Activation of MAPKs
svg
pdf and description
click for PDF MAPK (p38)
svg
pdf and description
click for PDF
MAPK and cell migration (JNK)
svg
pdf and description
click for PDF MAPK and cell migration (ERK)
svg
pdf and description
click for PDF MAPK and cell migration (P38)
svg
pdf and description
click for PDF
Pathway induced by RNA-virus
svg
pdf and description
click for PDF Pathway for general immune response
svg
pdf and description
click for PDF NFκB activation by viruses
svg
pdf and description
click for PDF
Viral interactions with nucleocytoplasmic transport pathways
svg
pdf and description
click for PDF Unfolded protein response within the endoplasmatic reticulum
svg
pdf and description
click for PDF Cell cycle during Epstein-Barr virus infection
svg
pdf and description
click for PDF
Herpes simplex infection
svg
pdf and description
click for PDF MyD88
svg
pdf and description
click for PDF CREB initiation
svg
pdf and description
click for PDF
NFκB
svg
pdf and description
click for PDF Focal adhesion
svg
pdf and description
click for PDF Actin pathway
svg
pdf and description
click for PDF
GADD34
svg
pdf and description
click for PDF DUSP
svg
pdf and description
click for PDF JAK/STAT
svg
pdf and description
click for PDF
Insulin
svg
pdf and description
click for PDF ARL13B
svg
pdf and description
click for PDF Endocytosis
svg
pdf and description
click for PDF
mTor
svg
pdf and description
click for PDF PI3K Akt
svg
pdf and description
click for PDF PI3K NFκB
svg
pdf and description
click for PDF
TGF-B interactions
svg
pdf and description
click for PDF RIP3/RIP1-mediated necrosis
svg
pdf and description
click for PDF Cell cycle, Peroxisome, Steroid biosynthesis, complement and coagulation cascades
svg
pdf and description
click for PDF
Endocytosis
svg
pdf and description
click for PDF Apoptosis
svg
pdf and description
click for PDF






ES 7: qRT-PCR

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qRT-PCR were performed for the human and bat homologous genes HAVCR1, NPC1 and TLR3. Please note that for the qRT-PCR new samples from an similar infection experiment of the same cell lines were used. We noticed that the infection rate was lower as in the cells used within the transcriptome study.

ES 7A: Primer Design

ES 7B: qRT-PCR values and 18S normalized mRNA abundances

Download EXCEL

ES 7C: Melting curves

NPC1 HAVCR1 TLR3



ES 7D: Agarose gel

2% agarose gel in TAE was run in 120V for 35 minutes. (-): control; water

On first gel, primer for bat HAVCR1 did not amplify. HAVCR1 was rerun on a second gel.





ES 7E: IFA

We repeated the infection of HuH7 and R06E-J cells and isolated RNA for qRT-PCR analyses. An IFA of these cells revealed that the infection rates were lower than in the previous experiment used for RNA-Seq.