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EBAME10: Functional enrichment in anvi'o

Amy D Willis

October 17 2025

ebame10_regression_pt1.Rmd

Background

This lab covers “functional enrichment” in anvi’o, in its simplest form.

The main learning objective is for you to deepen your understanding of regression models as tools to answer questions about your metagenomics data. See the lecture slides.

Functional enrichment is an example of estimating comparative parameters in metagenomics, so it gives us the chance to illustrate some concepts from lecture.

Functional enrichment fits broadly into the paradigm of pangenomics. If you want to learn about the amazing pangenomics tools in anvi’o, check out the tutorials at anvi’o.org/learn/.

Setup

Navigate to wherever you have been running anvi’o the last few days. Activate your anvi’o conda environment if needed. Confirm anvi’o commands run (e.g., anvi-migrate -h). If they don’t, fix it. These commands work on anvi’o v8 (or dev as of October 2025).

We’re going to work with data from the amazing (now a little outdated) pangenomics in anvi’o tutorial available here.

Download the zipped data, unzip it, and move into that directory.

wget https://ndownloader.figshare.com/files/28834476 -O Prochlorococcus_31_genomes.tar.gz
tar -zxvf Prochlorococcus_31_genomes.tar.gz
cd Prochlorococcus_31_genomes

Here we have some Prochlorococcus genomes in contigs DBs. How many genomes are there?

ls *.db | wc -l

Each was recovered from either a high-light ocean sample, or a low-light ocean sample. You can read about them in way more detail in the tutorial linked above.

We want to identify which COGs were present more often in one type of genomes. As discussed in the lecture, this is equivalent to asking whether the odds of detecting a COG in a genome differs based on where the genome was recovered from.

We actually don’t need to build a pangenome to answer this question*. All we need is

  1. A list of the genomes that we want to include in the analysis. Here, we’re going to use the external genomes file external-genomes.txt. (Check out its format with head external-genomes.txt.)
  2. The name of the file we want to save the results. I’m going to call it enrichment-output.txt.
  3. The annotation source. I’m going to choose COG14_FUNCTION.
  4. The groups we want to compare across. This info is contained in layer-additional-data.txt.

*You could, of course! It allows you to do lots of interesting things! It can take a while, though. The approach presented here is faster.

Attempting to run anvi-compute-functional-enrichment-across-genomes

One of the many cool things about anvi’o is that it’s hard to break stuff. So, as a general rule, I try things until my wish comes true. Often, the help messages are enough to get me there. Let’s see if they are enough today!

anvi-compute-functional-enrichment-across-genomes -e external-genomes.txt -o enrichment-output.txt --annotation-source COG14_FUNCTION

“error: the following arguments are required: -G/–groups-txt”. Ok, fine. That info is in layer-additional-data.txt:

head layer-additional-data.txt

Ok, maybe this will work?

anvi-compute-functional-enrichment-across-genomes -e external-genomes.txt -o enrichment-output.txt --annotation-source COG14_FUNCTION -G layer-additional-data.txt

“Config Error: A groups-txt file should have a column that is called group.”

Ok, cool! layer-additional-data.txt has what we need, but not in a column called group.

I hate remembering stuff, so of course, I asked my bff ChatGPT.

I have a file called layer-additional-data.txt. here's what it looks like 

head layer-additional-data.txt
isolate clade   light
AS9601  HL_II   HL
CCMP1375    LL_II   LL
EQPAC1  HL_I    HL
GP2 HL_II   HL
LG  LL_II   LL
MED4    HL_I    HL
MIT9107 HL_II   HL
MIT9116 HL_II   HL
MIT9123 HL_II   HL

I want to copy the column light and call it group. give me a cmd line one-liner thx

(You don’t have to say thanks, but maybe we’ll be less likely to turn into paperclips)

awk 'BEGIN{OFS="\t"} NR==1{$(NF+1)="group"} NR>1{$(NF+1)=$3}1' layer-additional-data.txt > layer-additional-data-with-group.txt

It created a new document with the additional column (Check with head layer-additional-data-with-group.txt)

Ok! Let’s try again!

anvi-compute-functional-enrichment-across-genomes -e external-genomes.txt -o enrichment-output.txt --annotation-source COG14_FUNCTION -G layer-additional-data-with-group.txt

“Config Error: The database at ‘Prochlorococcus_31_genomes/AS9601.db’ is outdated (this database is v20 and your anvi’o installation wants to work with v24). You can migrate your database without losing any data using the program anvi-migrate with either of the flags --migrate-safely or --migrate-quickly.”

Ok, so…

anvi-migrate *.db --migrate-safely

And now…

anvi-compute-functional-enrichment-across-genomes -e external-genomes.txt -o enrichment-output.txt --annotation-source COG14_FUNCTION -G layer-additional-data-with-group.txt

Hooray!!! Now let’s open up enrichment-output.txt and talk about it.

Can I just have the answer?

Ok :’)

wget https://ndownloader.figshare.com/files/28834476 -O Prochlorococcus_31_genomes.tar.gz
tar -zxvf Prochlorococcus_31_genomes.tar.gz
cd Prochlorococcus_31_genomes
awk 'BEGIN{OFS="\t"} NR==1{$(NF+1)="group"} NR>1{$(NF+1)=$3}1' layer-additional-data.txt > layer-additional-data-with-group.txt\n
anvi-migrate *.db --migrate-safely
anvi-compute-functional-enrichment-across-genomes -e external-genomes.txt -o enrichment-output.txt --annotation-source COG14_FUNCTION -G layer-additional-data-with-group.txt