Colonization of the N2 and CB4856 C. elegans strains with the CeMBio community at 120h post L1
Adrien Assie
Last update: June 09, 2020
Original code from P. Dirksen
| medium | time | bacteria | ID | type | DNA_batch | lms | |
|---|---|---|---|---|---|---|---|
| CB4856:6 | 0h : 2 | cembio:17 | 116-Cembio: 1 | Lawn : 3 | Min. :1 | 116-Cembio: 1 | |
| Lawn :3 | 120h:15 | 117-Cembio: 1 | Seed : 2 | 1st Qu.:1 | 117-Cembio: 1 | ||
| N2 :6 | 22-Cembio : 1 | worms:12 | Median :1 | 22-Cembio : 1 | |||
| Seed :2 | 23-Cembio : 1 | Mean :1 | 23-Cembio : 1 | ||||
| 24-Cembio : 1 | 3rd Qu.:1 | 24-Cembio : 1 | |||||
| 4-Cembio : 1 | Max. :1 | 4-Cembio : 1 | |||||
| (Other) :11 | (Other) :11 |
Check for obvious contaminants
Figure S3: Taxonomic composition of all samples on class level. Taxonomic composition is Gamma-, Alphaproteobacteria, and Bacteroidia, which is what we would expect based on the CeMBio strains. Few samples contain the remaining detected classes, which are most likely reagent contaminations. Sample 27 contains also Bacilli reads, which were not among the experimental strains, but among the mock community.
Identifying unresolved ASVs representing the CeMBio community
The total number of ASVs in the whole dataset is 195. However, only 16 ASVs are needed to account for 99.9% of all reads:
Figure S4: Cumulative proportion of reads. In total 16 ASVs account for 99.87% of all reads.
Merging ASVs belonging to the same CeMBio strain
Out of these 16 ASVs, 12 were identical in sequence to CeMBio 16S sequences, while 4 could not be resolved to strain level, likely due to sequencing artifacts despite denoising with dada2.
| No | Family | Genus | Species |
|---|---|---|---|
| 1 | Xanthomonadaceae | Stenotrophomonas | |
| 2 | Moraxellaceae | Acinetobacter | |
| 3 | Sphingobacteriaceae | Sphingobacterium | |
| 4 | Erwiniaceae | ||
| 5 | Enterobacteriaceae | ||
| 6 | Rhizobiaceae | Brucella | |
| 7 | Comamonadaceae | Limnohabitans | |
| 8 | Weeksellaceae | Chryseobacterium | |
| 9 | Pseudomonadaceae | Pseudomonas | |
| 10 | Pseudomonadaceae | Pseudomonas | |
| 11 |
In order to resolve these ambigous ASVs, BLAST analysis was performed against the genome-derived 16S sequences of the CeMBio strains. As a result, the ambigous ASV were assigned the following identities:
| query acc.ver | subject acc.ver | % identity | bit score | evalue |
|---|---|---|---|---|
| ASV001-Xanthomonadaceae-Stenotrophomonas- | JUb19 | 100 | 462 | 0 |
| ASV002-Moraxellaceae-Acinetobacter- | MYb10 | 100 | 462 | 0 |
| ASV003-Sphingobacteriaceae-Sphingobacterium- | BIGb0170 | 100 | 462 | 0 |
| ASV004-Erwiniaceae– | BIGb0393 | 100 | 462 | 0 |
| ASV005-Enterobacteriaceae– | CeEnt1 | 100 | 462 | 0 |
| ASV006-Rhizobiaceae-Brucella- | MYb71 | 100 | 462 | 0 |
| ASV007-Comamonadaceae-Limnohabitans- | BIGb0172 | 100 | 462 | 0 |
| ASV008-Weeksellaceae-Chryseobacterium- | JUb44 | 100 | 462 | 0 |
| ASV009-Pseudomonadaceae-Pseudomonas- | MSPm1 | 100 | 462 | 0 |
| ASV010-Pseudomonadaceae-Pseudomonas- | MYb11 | 100 | 462 | 0 |
| ASV011— | JUb134 | 100 | 462 | 0 |
Finally, the ASV belonging to the same organism were merged, leading to the following final ASVs:
| No | Family | Genus | Species | ASV |
|---|---|---|---|---|
| 1 | Xanthomonadaceae | Stenotrophomonas | JUb19 | ASV001 |
| 2 | Moraxellaceae | Acinetobacter | BIGb0170 | ASV002 |
| 3 | Sphingobacteriaceae | Sphingobacterium | MYb10 | ASV003 |
| 4 | Erwiniaceae | MYb71 | ASV004 | |
| 5 | Enterobacteriaceae | CEnt1, JUb66 | ASV005 | |
| 6 | Rhizobiaceae | Brucella | JUb44 | ASV006 |
| 7 | Comamonadaceae | Limnohabitans | BIGb0172 | ASV007 |
| 8 | Weeksellaceae | Chryseobacterium | BIGb0393 | ASV008 |
| 9 | Pseudomonadaceae | Pseudomonas | MYb11 | ASV009 |
| 10 | Pseudomonadaceae | Pseudomonas | MSPm1 | ASV010 |
| 11 | JUb134 | ASV011 |
Adjusting counts by rRNA copy number
We have all the genomes and information on rRNA gene number per strain. This data can be used to adjust the raw reads counts by gene copy number to enhance the estimate of relative cell counts.
| BIGb0170 | BIGb0172 | BIGb0393 | CEnt1, JUb66 | JUb19 | JUb44 |
|---|---|---|---|---|---|
| 7 | 6 | 7 | 15 | 4 | 7 |
| JUb134 | MYb10 | MYb11 | MYb71 | MSPm1 | NA |
|---|---|---|---|---|---|
| 3 | 7 | 5 | 4 | 4 |
Table S1: Taxonomic composition of worms raised on NGM and PFM agar plates.
|
|
##
## Pairwise comparisons using t tests with pooled SD
##
## data: estimate_richness(ps_exp)$InvSimpson and paste(sample_data(ps_exp)$med_type, sample_data(ps_exp)$time)
##
## CB4856 worms 120h Lawn Lawn 120h N2 worms 120h
## Lawn Lawn 120h 0.0112 - -
## N2 worms 120h 0.7972 0.0093 -
## Seed Seed 0h 3.2e-09 8.3e-08 3.2e-09
##
## P value adjustment method: fdr
Figure 1: Taxonomic composition of colonized C. elegans nematodes and plate lawns. (A) Proportion of CeMBio reads in the initial community assembly used as inoculum for the lawns. (B) Proportion of CeMBio reads in N2 and CB4856 nematodes and lawn samples. (C) Alpha diversity measures of mean observed no. of species (top) and Inverse Simpson Index (bottom) with standard deviation indicating richness and diversity of the communities.
Correlation of bacterial composition and load
Figure 2: Bacterial load of C. elegans nematodes colonized by the CeMBio community (A) Estimated CFU count in single nematodes. (B) Principle coordinate analysis of Bray Curtis distances. CFU-load, overlayed as a smooth response surface, is a significant predictor of the ordination result (Generalized additive model, df = 15, F = 2.523, p < 10^-5, R-square = 0.57)
##
## Family: gaussian
## Link function: identity
##
## Formula:
## y ~ poly(x1, 1) + poly(x2, 1)
##
## Parametric coefficients:
## Estimate Std. Error t value Pr(>|t|)
## (Intercept) 4.17214 0.07678 54.337 1.37e-05 ***
## poly(x1, 1) 0.04209 0.18808 0.224 0.8373
## poly(x2, 1) -0.44397 0.18808 -2.361 0.0994 .
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
##
##
## R-sq.(adj) = 0.42 Deviance explained = 65.2%
## -ML = -3.5912 Scale est. = 0.035374 n = 6Differential abundance of bacteria between worms and lawns
Beta diversity
Figure: Effect of medium and developmental time on microbiome diversity. A Differential abundance of the CeMBio strains in C. elegans nematodes compared to the plate lawns. Positive fold change indicates increased abundance in worms compared to lawns. Data was normalized and analysed using DESeq2. Filled points indicate fold changes significantly different from 0 for with a p < 0.01. B PCoA with Bray-Curtis distance. Ellipses denote lawn and worm samples, respectively.
Figure 2
Session information
## R version 3.5.3 (2019-03-11)
## Platform: x86_64-apple-darwin15.6.0 (64-bit)
## Running under: macOS Mojave 10.14.6
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## Matrix products: default
## BLAS: /Library/Frameworks/R.framework/Versions/3.5/Resources/lib/libRblas.0.dylib
## LAPACK: /Library/Frameworks/R.framework/Versions/3.5/Resources/lib/libRlapack.dylib
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## locale:
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## attached base packages:
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## other attached packages:
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