Microbial 'omics

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The Fecal Microbiota Transplantation study

Summary

The purpose of this page is to discuss key findings of the study “Adaptive ecological processes and metabolic independence drive microbial colonization and resilience in the human gut” by Watson et al.

On this page you will find data and ad hoc scripts for important analyses, results of which discussed in the paper.

If you have any questions and/or if you are unable to find an important piece of information here, please feel free to leave a comment down below, send an e-mail to us, or get in touch with us through Slack:

Study description

You can find a crude summary of our study here as a Twitter thread.

Briefly, our study involves the investigations of human gut metagenomes to understand the ecology of microbes before and after fecal microbiota transplantation (FMT) experiments, and the relevance of emerging ecological principals to inflammatory bowel disease (IBD). For this, we

• Reconstruct metagenome-assembled genomes (MAGs) from FMT donor gut metagenomes,
• Characterize the distribution of donor populations in donor and recipient gut metagenomes before and after FMT using these genomes,
• Investigate the prevalence of donor populations in global gut metagenomes,
• Define a set of quantitative criteria to determine ‘colonization’ events unambigously,
• Identify ‘high-fitness’ and ‘low-fitness’ donor populations based on their colonization abilities and prevalence in global gut metagenomes,
• Determine metabolic pathways that are enriched in either of these high-fitness or low-fitness groups,
• Study microbial genomes reconstructed from healthy individuals and individuals with IBD to test whether the enrichment of high-fitness and low-fitness genomes were identical between these cohorts (they were not).

Here is a sketch that aims to capture the points below:

Most of these steps are indeed detailed in our manuscript. On this page, you will find details of intermediate steps and anvi’o reusable data currencies to reproduce our main findings.

Reproducible donor and recipient metagenomes

doi:10.6084/m9.figshare.14331236 serves the anvi’o reproducible donor and recipient data.

This section will introduce the primary data objects that can be used in the anvi’o software ecosystem. This subsection will describe how they were generated, give access to them, and demonstrate how they can be used.

Background information

The NCBI BioProject accession ID PRJNA701961 gives access to raw sequences for each donor and recipient metagenome. Details of each metagenome and their individual accession IDs are listed in this supplementary table.

We generated our data objects from the shotgun sequencing of donor and recipient stool metagenomes. The program anvi-run-workflow took us from raw metagenomic sequences to anvi’o contigs and profile databases (which will be explained later) by running the anvi’o workflow for metagenomics in a scalable fashion. Details of this workflow is described in our study in detail, but briefly, for each donor and their recipients this workflow, co-assembled donor metagenomes, profiled resulting donor contigs in the context of donor and recipient metagenomes through metagenomic read recruitment (which enabled us to reconstruct and refine donor genomes using anvi-interactive and anvi-refine, and summarized the coverage and detection of donor genomes across metagenomes for downstream analyses.

We uploaded the resulting data packs to FigShare and the following subsections explain how to download and work with them.

You can download either or both data packs for donor A (4.14 Gb compressed) and donor B (4.59 Gb compressed) the following way:

# download donor A data pack:
-o FMT_DONOR_A_AND_RECIPIENTS.tar.gz

-o FMT_DONOR_B_AND_RECIPIENTS.tar.xz


Once you download either (or both) of these data packs, the following commands will enable you to unpack and investigate their contents (the example here is given for donor A, but will also work for donor B with slight changes):

# unpack (this should take about 30 seconds on a standard
# laptop and result in a directory that takes about 8Gb
# of storage space
tar -zxvf FMT_DONOR_A_AND_RECIPIENTS.tar.gz

# go into the directory:
cd FMT_DONOR_A_AND_RECIPIENTS

# make sure you have all the files:
ls -l

total 7.8G
4.0G Mar 28 11:19 AUXILIARY-DATA.db
1.4G Mar 28 11:18 CONTIGS.db
4.4K Mar 28 11:19 MAGs-order-by-detection.newick
2.5G Mar 28 11:38 PROFILE.db


If you see a similar output, you have everything you need to reproduce and scrutinize our figures and findings interactively.

These files give access to (1) the co-assemblies of the stool metagenomes from a given donor, (2) gene calls and annotations of gene functions for each donor contig, (3) nucleotide-resolved coverage and detection data for each donor contig across all donor and recipient metagenomes, and (4) genomes we have reconstructed from these data (which is stored as a collection named default in the merged profile database).

Reproduce

The two main files in these data pack are CONTIGS.db and PROFILE.db, and the vast majority of analyses in our study used the information stored in these artifacts.

Examples below are for donor A, but will also work for donor B with slight changes.

Genomes described in our study is stored in a collection called default:

anvi-show-collections-and-bins -p PROFILE.db

Collection: "default"
===============================================
Collection ID ................................: default
Number of bins ...............................: 128
Number of splits described ...................: 21,741
Bin names ....................................: DA_MAG_00001, DA_MAG_00002, DA_MAG_00003, DA_MAG_00004, DA_MAG_00005, DA_MAG_00006, DA_MAG_00007, DA_MAG_00008, DA_MAG_00009, DA_MAG_00010, DA_MAG_00011,
DA_MAG_00012, DA_MAG_00013, DA_MAG_00014, DA_MAG_00015, DA_MAG_00016, DA_MAG_00017, DA_MAG_00018, DA_MAG_00019, DA_MAG_00020, DA_MAG_00021, DA_MAG_00022,
DA_MAG_00023, DA_MAG_00024, DA_MAG_00025, DA_MAG_00026, DA_MAG_00027, DA_MAG_00028, DA_MAG_00029, DA_MAG_00030, DA_MAG_00031, DA_MAG_00032, DA_MAG_00033,
DA_MAG_00034, DA_MAG_00035, DA_MAG_00036, DA_MAG_00037, DA_MAG_00038, DA_MAG_00039, DA_MAG_00040, DA_MAG_00041, DA_MAG_00042, DA_MAG_00043, DA_MAG_00044,
DA_MAG_00045, DA_MAG_00046, DA_MAG_00047, DA_MAG_00048, DA_MAG_00049, DA_MAG_00050, DA_MAG_00051, DA_MAG_00052, DA_MAG_00053, DA_MAG_00054, DA_MAG_00055,
DA_MAG_00056, DA_MAG_00057, DA_MAG_00058, DA_MAG_00059, DA_MAG_00060, DA_MAG_00061, DA_MAG_00062, DA_MAG_00063, DA_MAG_00064, DA_MAG_00065, DA_MAG_00066,
DA_MAG_00067, DA_MAG_00068, DA_MAG_00069, DA_MAG_00070, DA_MAG_00071, DA_MAG_00072, DA_MAG_00073, DA_MAG_00074, DA_MAG_00075, DA_MAG_00076, DA_MAG_00077,
DA_MAG_00078, DA_MAG_00079, DA_MAG_00080, DA_MAG_00081, DA_MAG_00082, DA_MAG_00083, DA_MAG_00084, DA_MAG_00085, DA_MAG_00086, DA_MAG_00087, DA_MAG_00088,
DA_MAG_00089, DA_MAG_00090, DA_MAG_00091, DA_MAG_00092, DA_MAG_00093, DA_MAG_00094, DA_MAG_00095, DA_MAG_00096, DA_MAG_00097, DA_MAG_00098, DA_MAG_00099,
DA_MAG_00100, DA_MAG_00101, DA_MAG_00102, DA_MAG_00103, DA_MAG_00104, DA_MAG_00105, DA_MAG_00106, DA_MAG_00107, DA_MAG_00108, DA_MAG_00109, DA_MAG_00110,
DA_MAG_00111, DA_MAG_00112, DA_MAG_00113, DA_MAG_00114, DA_MAG_00115, DA_MAG_00116, DA_MAG_00117, DA_MAG_00118, DA_MAG_00119, DA_MAG_00120, DA_MAG_00121,
DA_MAG_00122, DA_MAG_00123, DA_MAG_00124, DA_MAG_00125, DA_MAG_00126, DA_MAG_00127, DA_MAG_00128



The genome names shown here will indeed match to the genome names in our supplementary tables.

To interactively visualize all donor genomes stored in the collection we called default across all donor and recipient metagenomes, you can run this anvi-interactive command:

anvi-interactive --profile-db PROFILE.db \
--contigs-db CONTIGS.db \
--collection-name default \
--tree MAGs-order-by-detection.newick


Which will open a browser window to display the detection of donor A genomes which reproduces the panel in Figure 01 interactively:

If you are interested in investigating the coverages of a single donor genome, say DA_MAG_00052, you can run the following anvi-refine command:

anvi-refine --profile-db PROFILE.db \
--contigs-db CONTIGS.db \
--collection-name default \
--bin-id DA_MAG_00052


Which will open a browser window to display the detection of each contig in the donor A genome DA_MAG_00052:

Where you can right-click on any split and inspect nucleotide-level coverage values, genes and their functions for each section of the genome.

In addition to the examples shown above, these data packs can be used for additional investigations thanks to their versatility. CONTIGS.db is an anvi’o contigs database, and any anvi’o program that runs on contigs databases will run on this file. Similarly, PROFILE.db is an anvi’o profile database, and you can find here a list of programs in the anvi’o ecosystem that will work with this artifact.

Estimating the global prevalence of donor genomes

Details of each publicly available metagenome used and their individual accession IDs are listed in this supplementary table.

To determine the prevalence of donor genomes in 17 different countries, we ran a separate anvi’o metagenomics workflow. This time, the workflow (1) recruited reads from 1,984 publicly available gut metagenomes to our donor contigs using bowtie2, (2) profiled mapping results and (3) summarized the detection of each MAG in each global metagenome.

Included in the resulting summary files is information about the detection of each donor genome in each global metagenome. We summarized the detection results by country, using a minimum detection threshold of 0.25. You can reproduce this step the following way:

# download the input files for donor A (3.7Mb) and donor B (5.2Mb)
for file in detection-global-DA.txt \
detection-global-DB.txt;
do curl -L https://merenlab.org/data/fmt-gut-colonization/files/${file} \ -o${file};
done

curl -L https://merenlab.org/data/fmt-gut-colonization/files/summarize-global-detection.py \
-o summarize-global-detection.py

# run the script and look at the output files:
# detection-global-by-country-DA.txt and
# detection-global-by-country-DB.txt
python3 summarize-global-detection.py


Investigating the ecological forces driving colonization outcomes

Recreating input data files

The following analyses use data tables derived from the output of anvi-summarize. You can download the tables directly using the instructions in this section, or you can reproduce them yourself using the CONTIGS.db and PROFILE.db contained in the donor A and donor B data packs like so:

To fully reproduce what we did, you need to run anvi-summarize with the --init-gene-coverages flag. Andrea crashed her laptop when testing this, so it is recommended that you only do this if you have a fancy, powerful computer with sufficient resources. :(

# make sure you're in the directory containing the data packs
ls -l

Apr  1 12:26 FMT_DONOR_A_AND_RECIPIENTS
Mar 31 17:17 FMT_DONOR_A_AND_RECIPIENTS.tar.gz
Apr  1 12:30 FMT_DONOR_B_AND_RECIPIENTS
Mar 31 18:34 FMT_DONOR_B_AND_RECIPIENTS.tar.gx

# run anvi-summarize
anvi-summarize --pan-or-profile-db FMT_DONOR_A_AND_RECIPIENTS/PROFILE.db \
--contigs-db FMT_DONOR_A_AND_RECIPIENTS/CONTIGS.db \
--collection-name default \
--init-gene-coverages \
--output-dir FMT_DONOR_A_AND_RECIPIENTS/SUMMARY

# repeat above step for donor B

curl -L https://merenlab.org/data/fmt-gut-colonization/files/make-scg-cov-tables.py \
-o make-scg-cov-tables.py

# run script and note the output files:
# scg-cov-DA.txt and
# scg-cov-DB.txt
python3 make-scg-cov-tables.py

# copy other relevant files to working directory to follow the rest of the workflow
cp FMT_DONOR_A_AND_RECIPIENTS/SUMMARY/bins_across_samples/detection.txt detection-FMT-DA.txt
cp FMT_DONOR_B_AND_RECIPIENTS/SUMMARY/bins_across_samples/detection.txt detection-FMT-DB.txt

cp FMT_DONOR_A_AND_RECIPIENTS/SUMMARY/bins_across_samples/mean_coverage_Q2Q3.txt mean-cov-DA.txt
cp FMT_DONOR_B_AND_RECIPIENTS/SUMMARY/bins_across_samples/mean_coverage_Q2Q3.txt mean-cov-DB.txt


This section will describe how to recreate Figure 2 in our study:

We wanted to determine whether colonization outcomes were driven primarily by neutral or adaptive ecological forces. If colonization outcomes were driven by neutral forces, we would expect the successful colonizers to be the populations that were the most abundant in the donor stool sample used for transplantation. These would be the populations most likely to survive the bottleneck event that occured when a portion of the stool sample was selected for transplantation, and the most likely to survive the effects of ecological drift in the gut. Alternatively, if colonization outcomes were driven by adaptive ecological forces, we would expect the successful colonizers to be the populations that were the most fit in the gut environment.

To test whether colonization outcomes were driven by neutral or adaptive forces we measured the correlation of dose and fitness with colonization outcome. We measured dose using the mean coverage of a population in the donor stool sample used for transplantation and we used population prevalence in healthy adult guts as a proxy for fitness.

Preliminary analysis

As a simple preliminary analysis we first measured whether the fitness of a population was correlated with its detection in donor or recipient post-FMT metagenomes, to see if the post-FMT gut environment was selecting for fitter populations than the donor gut environment.

If you want to reproduce this analysis, you can do so with the following steps:

# download the input files
detection-FMT-DA.txt \
detection-FMT-DB.txt \
detection-global-by-country-DA.txt \
detection-global-by-country-DB.txt;
do curl -L https://merenlab.org/data/fmt-gut-colonization/files/${file} \ -o${file};
done

# make sure you have all the files
ls -l

total 456
46K Mar 31 12:30 detection-FMT-DA.txt
58K Mar 31 12:30 detection-FMT-DB.txt
42K Mar 31 12:30 detection-global-by-country-DA.txt
60K Mar 31 12:30 detection-global-by-country-DB.txt

curl -L https://merenlab.org/data/fmt-gut-colonization/files/make-mean-detec-vs-prev-table.py \
-o make-mean-detec-vs-prev-table.py

# run the script to make aforementioned tables
python3 make-mean-detec-vs-prev-table.py

curl -L https://merenlab.org/data/fmt-gut-colonization/files/Figure-02A.R \
-o Figure-02A.R

# run the script to visualize results
Rscript Figure-02A.R


Which generates a PDF that looks like this:

Defining colonization success and failure

Our preliminary results indicated that FMT may be selecting for fitter microbes in the recipient post-FMT gut environment. However, we wanted to more directly test whether neutral or adaptive forces were driving colonization.

We began with a simple definition of colonization: a population is absent in a recipient pre-FMT, present in the donor stool samples used for transplantation, and present in the recipient post-FMT. Whereas if a population is present in the donor stool sample used for transplantation and absent in the recipient pre-FMT, it failed to colonize. However, we decided that looking at the presence or absence of population genomes in donor and recipient samples did not provide sufficient resolution. If a population is present in the donor and in the recipient pre-FMT, how do we know if the post-FMT population is native to the recipient or came from the donor? Even if we don’t see a population pre-FMT, how do we know that the post-FMT population wasn’t already present in the recipient at undetectable levels? To resolve these ambiguities, we used subpopulation, or strain, level information from DESMAN. DESMAN provides the number of subpopulations, or strains, of each population in each metagenome, along with their relative abundances.

This led us to create the definition of colonization, or non-colonization, outlined in Supplementary Figure 4:

To run the above workflow to determine if a population colonized a recipient in our study, you can do the following:

# download additional input files
for file in scg-cov-DA.txt \
scg-cov-DB.txt \
subpop-comp-DA.txt \
subpop-comp-DB.txt \
subpop-num-DA.txt \
subpop-num-DB.txt;
do curl -L https://merenlab.org/data/fmt-gut-colonization/files/${file} \ -o${file};
done

curl -L https://merenlab.org/data/fmt-gut-colonization/files/determine-colonization.py \
-o determine-colonization.py

# run the script and look at the output files:
# colonized-DA.txt,
# colonized-DB.txt,
# did-not-colonize-DA.txt and
# did-not-colonize-DB.txt
python3 determine-colonization.py


We then used the output of this workflow to measure the correlation of dose and fitness with colonization outcome, to determine whether colonization is driven by neutral or adaptive forces.

Correlation of dose and fitness with colonization outcome

We used logistic regression to test for a correlation between dose and colonization outcome, and fitnes and colonization outcome. We then further evaluated our regression models using ROC curves.

If you would like to recreate this analysis, you can do so through the following steps:

# download additional files
for file in mean-cov-DA.txt \
mean-cov-DB.txt;
do curl -L https://merenlab.org/data/fmt-gut-colonization/files/${file} \ -o${file};
done

curl -L https://merenlab.org/data/fmt-gut-colonization/files/make-summary-tables-for-regression.py \
-o make-summary-tables-for-regression.py

# run script to consolidate data
python3 make-summary-tables-for-regression.py

curl -L https://merenlab.org/data/fmt-gut-colonization/files/Figure-02BC.R \
-o Figure-02BC.R

# run script to visualize results
Rscript Figure-02BC.R


Which generates a PDF that looks like this:

Correlation of fitness with dose

Our logistic regression analysis showed that fitness was correlated with colonization success in the donor B cohort, and dose was not. This indicates that within the donor B cohort, colonization outcome was likely driven by adaptive, rather than neutral, ecological forces.

However, within the donor A cohort both fitness and dose were correlated with colonization success. The donor A ROC curves suggested a possible correlation between fitness and dose, confounding our ability to determine which of these factors were relevant to colonization outcome. To see if this was the case we performed a linear regression to see if fitness and dose were correlated with one another within each cohort.

To reproduce that analysis, you can follow these steps:

# download the script
curl -L https://merenlab.org/data/fmt-gut-colonization/files/Figure-02D.txt \
-o Figure-02D.R

# run the script
Rscript Figure-02D.R


Which produces a PDF that looks like this:

Indeed, in donor A populations fitness and dose are correlated with one another, while this is not the case for donor B populations.

Investigating metabolic competence among microbial genomes reconstructed from healthy individuals and individuals with IBD

doi:10.6084/m9.figshare.14225840 serves each genome used in the analysis below.

This section will describe how to go from FASTA files for a given set of genomes to the Figure 4 in our study:

Our analyses in the previous chapters of our study showed that while the healthy donor environment could support both high-fitness and low-fitness populations, challenging microbes to colonize a new environment or to withstand massive ecosystem perturbation during FMT selects for high-fitness populations, suggesting that metabolic competence is a more critical determinant of fitness during stress than during homeostasis. Based on these observations, we hypothesized that,

• A gut environment in homeostasis will support a range of microbial populations with a wide spectrum of metabolic competency, and
• A gut environment under stress will select for high metabolic competency in microbial populations.

Publicly available datasets

To test these hypotheses, we proceeded to compared genomes reconstructed from a cohort of healthy individuals by Pasolli et al. (2019) to genomes reconstructed from individuals who were diagnosed with inflammatory bowel disease (IBD). Our IBD dataset was composed of two cohorts: a set of patients with pouchitis from Vineis et al (2016), and a set of pediatric Crohn’s disease patients described in Quince et al. (2015).

FASTA files to anvi’o contigs databases

At this stage we have multiple FASTA files for each individual in three cohorts (healthy, pouchitis, and Crohn’s) for downstream analyses. We first converted each FASTA file into an anvi’o contigs-db using the program anvi-gen-contigs-database and then processed each resulting contigs-db using,

• The program anvi-run-hmms to run hidden Markov models for the identification of single-copy core genes,
• The program anvi-run-scg-taxonomy to identify and store taxonomic information,
• and the program anvi-run-kegg-kofams to identify KEGG orthologs in each of our genomes for metabolic reconstruction.

The resulting contigs databases are publicly available. If you would like to reproduce the steps below, you should first download the following file [943 Mb] to your work directory,

curl -L https://ndownloader.figshare.com/files/26842652 \
-o WATSON_ET_AL_CONTIGS_DBS_FOR_METABOLIC_COMPARISON.tar.gz


And unpack it [2.7 Gb]:

tar -zxvf WATSON_ET_AL_CONTIGS_DBS_FOR_METABOLIC_COMPARISON.tar.gz


After having generated a contigs database for each genome, we used all the information about these genomes that were available to us to prepared an external-genomes file. This is simply a TAB-delimited text file that describes the location of each contigs database for each genome on the disk, along with optional metadata columns to describe their properties. Here are a few lines from this file:

name individual cohort from accession ID genome_completion genome_redundancy num_splits total length t_phylum t_class t_order t_family t_genus t_species contigs_db_path
POU_P204_MAG_00001 POU_P204 POUCHITIS Vineis et al. (doi:10.1128/mBio.01713-16) phs000262.v3.p2 (dbGaP) 94.37 0.00 104 2081795 Firmicutes Clostridia Oscillospirales Acutalibacteraceae UMGS1071 None WATSON_ET_AL_CONTIGS_DBS_FOR_METABOLIC_COMPARISON/POU_P204_MAG_00001.db
POU_P204_MAG_00002 POU_P204 POUCHITIS Vineis et al. (doi:10.1128/mBio.01713-16) phs000262.v3.p2 (dbGaP) 100.00 0.00 129 2564734 Firmicutes Clostridia Lachnospirales Anaerotignaceae Anaerotignum Anaerotignum sp001304995 WATSON_ET_AL_CONTIGS_DBS_FOR_METABOLIC_COMPARISON/POU_P204_MAG_00002.db
POU_P204_MAG_00003 POU_P204 POUCHITIS Vineis et al. (doi:10.1128/mBio.01713-16) phs000262.v3.p2 (dbGaP) 98.59 1.41 202 4105023 Bacteroidota Bacteroidia Bacteroidales Bacteroidaceae Bacteroides Bacteroides fragilis WATSON_ET_AL_CONTIGS_DBS_FOR_METABOLIC_COMPARISON/POU_P204_MAG_00003.db
POU_P204_MAG_00004 POU_P204 POUCHITIS Vineis et al. (doi:10.1128/mBio.01713-16) phs000262.v3.p2 (dbGaP) 98.59 5.63 263 5146152 Bacteroidota Bacteroidia Bacteroidales Bacteroidaceae Bacteroides None WATSON_ET_AL_CONTIGS_DBS_FOR_METABOLIC_COMPARISON/POU_P204_MAG_00004.db
(…) (…) (…) (…) (…) (…) (…) (…) (…) (…) (…) (…) (…) (…) (…) (…)
HMP_1061_SRS011061_bin.13 HMP_1061 HEALTHY Passoli et al. (doi:10.1016/j.cell.2019.01.001) SRS011061 76.06 40.85 233 1846034 Bacteroidota Bacteroidia Bacteroidales Rikenellaceae Alistipes Alistipes putredinis WATSON_ET_AL_CONTIGS_DBS_FOR_METABOLIC_COMPARISON/HMP_2012__SRS011061__bin.13.db
HMP_1061_SRS011061_bin.14 HMP_1061 HEALTHY Passoli et al. (doi:10.1016/j.cell.2019.01.001) SRS011061 98.59 0.00 219 2831497 Firmicutes Clostridia Oscillospirales Ruminococcaceae CAG-353 CAG-353 sp900066885 WATSON_ET_AL_CONTIGS_DBS_FOR_METABOLIC_COMPARISON/HMP_2012__SRS011061__bin.14.db
HMP_1061_SRS011061_bin.20 HMP_1061 HEALTHY Passoli et al. (doi:10.1016/j.cell.2019.01.001) SRS011061 85.92 0.00 300 1255151 Firmicutes Clostridia TANB77 CAG-508 CAG-273 CAG-273 sp000435755 WATSON_ET_AL_CONTIGS_DBS_FOR_METABOLIC_COMPARISON/HMP_2012__SRS011061__bin.20.db
HMP_1061_SRS011061_bin.23 HMP_1061 HEALTHY Passoli et al. (doi:10.1016/j.cell.2019.01.001) SRS011061 77.46 4.23 391 1510418 Firmicutes Clostridia Oscillospirales Acutalibacteraceae UMGS172 UMGS172 sp900539855 WATSON_ET_AL_CONTIGS_DBS_FOR_METABOLIC_COMPARISON/HMP_2012__SRS011061__bin.23.db
HMP_1061_SRS011061_bin.26 HMP_1061 HEALTHY Passoli et al. (doi:10.1016/j.cell.2019.01.001) SRS011061 100.00 2.82 232 1903353 Firmicutes Negativicutes Acidaminococcales Acidaminococcaceae Succiniclasticum Succiniclasticum sp900544275 WATSON_ET_AL_CONTIGS_DBS_FOR_METABOLIC_COMPARISON/HMP_2012__SRS011061__bin.26.db
(…) (…) (…) (…) (…) (…) (…) (…) (…) (…) (…) (…) (…) (…) (…) (…)

You can also download a copy of it on your disk and explore it the following way:

curl -L https://merenlab.org/data/fmt-gut-colonization/files/external-genomes.txt \
-o external-genomes.txt


The next step is to estimate the completion of each metabolic module described in KEGG database for each genome using the program anvi-estimate-metabolism:

Computing metabolic completion

anvi-estimate-metabolism -e external-genomes.txt \
--matrix-format \
-O metabolism


On a laptop computer this command will take up-to 25 minutes to characterize all metabolic modules in 604 genomes, and will generate the following files:

• metabolism-completeness-MATRIX.txt
• metabolism-ko_hits-MATRIX.txt
• metabolism-presence-MATRIX.txt

The output file we will use for downstream analyses is metabolism-completeness-MATRIX.txt (and if you don’t want to wait for the anvi-estimate-metabolism, you can download this file into your work directory from here). In this particular file each column represents a genome, each row represents a KEGG module, and each data point is the estimated completeness of a given KEGG module in a given genome. Here is a few lines and columns from this file:

module POU_P204_MAG_00001 POU_P204_MAG_00002 POU_P204_MAG_00003 POU_P204_MAG_00004 POU_P204_MAG_00005 (…)
M00001 0.8 0.9 1.0 1.0 0.9 (…)
M00002 0.66 1.0 1.0 1.0 1.0 (…)
M00003 0.87 0.87 1.0 1.0 0.75 (…)
M00004 0.57 0.71 1.0 1.0 1.0 (…)
M00005 1.0 1.0 1.0 1.0 1.0 (…)
M00006 0.0 0.0 1.0 1.0 1.0 (…)
M00007 0.75 1.0 1.0 1.0 1.0 (…)
M00008 0.0 0.0 0.75 0.75 0.5 (…)
M00009 0.25 0.5 0.62 0.75 0.56 (…)
(…) (…) (…) (…) (…) (…) (…)

Visualizing data in R

The rest of our analysis will take place in R. You can download the entire R script we used to generate relevant figure, and run it in your work directory get all the figures in a single step the following way:

# download the script
curl -L https://merenlab.org/data/fmt-gut-colonization/files/plot-metabolic-competency.R \
-o plot-metabolic-competency.R

# turn the executable bit on
chmod +x plot-metabolic-competency.R

# run it, and investigate the resulting PDF files:
./plot-metabolic-competency.R


The following steps highlight and describe some of the relevant steps in the script. As a reminder, we will need the following libraries to be initialized for everything downstream to run:

#!/usr/bin/env Rscript
library(ggplot2)
library(MASS)
library(ggridges)
library(gridExtra)
library(reshape2)
library(reshape)


We first read in the metabolic completion and external genomes files:

module_completion <- read.table(file='metabolism-completeness-MATRIX.txt', header = TRUE, sep = "\t")


A quick check to make sure the number of genomes per group makes sense based on what we know about our groups:

   CROHNS  FMT_HIGH_FITNESS  FMT_LOW_FITNESS   HEALTHY    POUCHITIS
256                20               20       264           44


We then turn the matrix formatted file into a data frame, and add some relevant information per genome using the external genomes file:

# turn the boring matrix format into a data frame
df <- melt(module_completion)

# set some meaningful column names
colnames(df) <- c('module', 'genome', 'completion')

# use the external genomes file to associate each genome with a 'group',
# and an individual:
df$group <- external_genomes$cohort[match(df$genome, external_genomes$name)]
df$individual <- external_genomes$individual[match(df$genome, external_genomes$name)]


Our matrix contains 303 modules. But our statistical enrichment analysis of the high-fitness versus low-fitness genomes had revealed a set of 33 modules. Thus, we want to investigate only those modules across these new genomes to investigate whether there is a difference in genomes across cohorts:

df <- df[df$module %in% modules_of_interest, ]  Finally, some boring steps of defining explicit orders for x-axes in the visualizations that will follow: # some boring steps of defining explicit orders for x-axes in boxplots HEALTHY_subset <- aggregate(df[df$group == "HEALTHY", 3], list(df[df$group == "HEALTHY", ]$individual), median)
HEALTHY_order <- HEALTHY_subset[order(-HEALTHY_subset$x),]$Group.1
POUCHITIS_subset <- aggregate(df[df$group == "POUCHITIS", 3], list( df[df$group == "POUCHITIS", ]$individual), median) POUCHITIS_order <- POUCHITIS_subset[order(-POUCHITIS_subset$x),]$Group.1 CROHNS_subset <- aggregate(df[df$group == "CROHNS", 3], list( df[df$group == "CROHNS", ]$individual), median)
CROHNS_order <- CROHNS_subset[order(-CROHNS_subset$x), ]$Group.1
individuals_order <- c(c('FMT_HIGH_FITNESS', 'FMT_LOW_FITNESS'), HEALTHY_order, POUCHITIS_order, CROHNS_order)

# set explicit group orders, and assign some group colors
groups_order <- c("FMT_HIGH_FITNESS", "FMT_LOW_FITNESS", "HEALTHY", "POUCHITIS", "CROHNS")
group_colors <- c("#ec5f1c", "#034f84", "#feb236", "#86af49", "#ff0202")


After these preparations, we are ready to run the following section,

# plot the boxplots
pdf(file = "boxplots.pdf",  width = 13, height = 5)
plot_individuals <- ggplot(data=df, aes(x=individual, y=completion, group=individual)) +
geom_boxplot(aes(fill=group), alpha=0.35, outlier.shape = NA, color='#808080') +
geom_jitter(colour='#222222', width = 0.20, height = 0.02, size=0.1, alpha=0.5) +
theme_bw() +
theme(legend.position="bottom", axis.title.y = element_blank(), axis.text.y = element_blank(), axis.ticks.y = element_blank()) +
xlab("Individuals") +
scale_x_discrete(limits = individuals_order) +
scale_fill_manual(values = group_colors)

plot_groups <- ggplot(data=df, aes(x=group, y=completion, group=group)) +
geom_boxplot(aes(fill=group), alpha=0.35, outlier.shape = NA, color=NA) +
geom_violin(fill="#505050", alpha=0.35, width=1.3, colour = '#505050') +
geom_jitter(colour='#222222', width = 0.3, height = 0.02, size=0.1, alpha=0.05) +
theme_bw() +
theme(legend.position="bottom") +
ylab("Metabolic module completion") +
xlab("Groups") +
scale_x_discrete(limits = groups_order) +
scale_fill_manual(values = group_colors)

grid.arrange(plot_groups, plot_individuals, ncol=2, widths=c(1, 3))
dev.off()


which generates a PDF file in our work directory that looks like this:

To visualize the ridge-line plots, we first selected a set of modules that differed significantly between the genomes from the healthy cohort and from those who were diagnosed with IBD to show some examples:

# modules_to_display <- c("M00924","M00122", "M00023", "M00028", "M00570", "M00082", "M00844", "M00015",  "M00526", "M00022")
dfx <- df[df$module %in% modules_to_display, ] dfx$module = factor(dfx$module, levels=modules_to_display)  Then, running the following section, pdf(file = "ridges-for-metabolisms.pdf", width = 13, height = 5) ggplot(data=dfx, aes(x = completion, y = individual, fill = group)) + geom_density_ridges(scale = 10, size = 0.25, rel_min_height = 0.01, alpha=0.35, colour="#222222") + theme_ridges() + scale_y_discrete(limits = individuals_orderx) + scale_fill_manual(values = group_colors) + scale_x_continuous(breaks=c(0, 1)) + coord_cartesian(xlim = c(-0.5, 1.5)) + theme(legend.position="bottom", axis.title.y = element_blank(), axis.text.y = element_blank(), axis.ticks.y = element_blank()) + facet_wrap(. ~ module, nrow = 2) dev.off()  which generates a PDF file in our work directory that looks like this: Finally we run the following test to estimate the statistical significance of differences to report in our study, printf <- function(...) invisible(print(sprintf(...))) printf("DIFFERENCES BETWEEN HEALTHY vs CROHNS + POUCHITIS for RIDGELINE PLOTS") for(module in modules_to_display){ w <- wilcox.test(dfx[dfx$group == "HEALTHY" & dfx$module == module, ]$completion, dfx[dfx$group %in% c("CROHNS", "POUCHITIS") & dfx$module == module, ]$completion) printf("%s: %f", module, w$p.value)
}


And combined both panels in Inkscape to finalize the figure for publication.