Microbial 'omics


This article describes various ways to import gene-level taxonomy into anvi’o (i.e., you have taxonomical annotations for each gene, and you want them to be in your database). Which can be very useful during the manual curation of metagenome-assembled genomes.

This post describes ways to import gene-level taxonomy data. We have some parsers to make sense of the output of some programs, but you can also import raw taxonomy for your genes you curated. If what you find here does not solve your problem, please feel free to suggest new ways to deal with gene-level taxonomy, by entering an issue.

The basic workflow goes like this: (1) generate your contigs database from your FASTA file, (2) export your gene sequences, (3) annotate them with taxonomy, and (4) import results back into your contigs database using anvi-import-taxonomy program.

Important note: There are many ways to have your genes annotated with taxonomy. But, there is only one way to make sure the gene IDs in your taxonomy files correspond to the gene caller IDs in the database: export your DNA or AA sequences from the anvi’o contigs database you wish to annotate using the anvi’o program anvi-get-sequences-for-gene-calls.

Table of Contents

Kaiju

Jarrod J. Scott, very kindly helped us to develop a parser for Kaiju and add it in anvi’o, and kindly provided the following tutorial to demonstrate how to import Kaiju output into anvi’o. We thank him very much for his patience and help throughout this entire process.

Kaiju parser will be available with anvi’o v5.

Anvi’o has a parser for the Kaiju classifier. The approach is described in in Menzel et al., “Fast and sensitive taxonomic classification for metagenomics with Kaiju” (open access). GitHub page for Kaiju includes detailed instructions on installing Kaiju, as well as obtaining and formatting databases.

Assuming you already have an anvi’o contigs database with gene calls, getting the file(s) for Kaiju is easy. Simply run:

anvi-get-sequences-for-gene-calls -c CONTIGS.db -o gene_calls.fa

Sweet, you are now ready to run Kaiju on your FASTA file!

The Kaiju package comes with many handy scripts for analyzing the taxonomic content of you samples. For the purpose of the anvi’o parser and getting the Kaiju output into you contigs database you will need to run three scripts:

  • First you need to run makeDB.sh to create a reference database.

  • Next, run the Kaiju command to classify you genes.

  • Finally, run addTaxonNames to amend the Kaiju output with taxon names.

First, download and format a reference database. You only need to run this once unless you want multiple databases or you want to update an existing database.

Kaiju offers four main databases: NCBI’s RefSeq, proGenomes, NCBI’s nr, and the Marine Metagenomic Portal. See the Kaiju documentation, or simply type makeDB.sh -h for a complete, most up-to-date list.

The following command will download and format the NCBI’s non-redundant protein database ‘nr’ with the addition of fungi and microbial eukaryotes using 20 parallel threads:

makeDB.sh -e -t 20

This is the largest database offered by Kaiju, and once downloaded and formatted it will take about 65 Gb space on your disk. In contrast, proGenome and the Marine Portal database takes up 13 Gb and 8 Gb, respectively. See the Kaiju GitHub page for detailed computational requirements for each database.

Choose a suitable directory to house your database. Once database downloading and formatting is complete, Kaiju only needs the files kaiju_db_nr_euk.fmi, nodes.dmp, and names.dmp. You can erase everything else including the genome directory, as well as the *.bwt and *.sa files.

Remember that you only need to run makeDB.sh once, and now you are done with it.

Now the database is ready, its time to do some classifying. Kaiju offers many options to tweak your classification parameters. You can explore these further by typing kaiju -h or checking out the documentation. For the purposes of the anvi’o parser however, you must, must, must enable the verbose output with the -v flag. The parser expects a defined number of columns which are obtained with the -v flag

To run the classifier, Kaiju needs the location of the nodes.dmp file, the database file (.fmi), your FASTA file, and an output filename (PLUS the -v option). Here is an example:

kaiju -t /path/to/nodes.dmp \
      -f /path/to/kaiju_db.fmi \
      -i gene_calls.fa \
      -o gene_calls_nr.out \
      -z 16 \
      -v

The -z option indicates the number of parallel threads. For reference, on my computer, ~250,000 genes took about 3 minutes with 16 threads and 5GB of RAM (80GB total) plus another ~15 minutes to read and load the db.

The output is a 7-column TAB-delimited file that contains (1) whether the read is classified (C) or unclassified (U), (2) the read ID, (3) NCBI taxon ID of closest hit, (4) best match score, (5) comma-separated list of taxon IDs with best match, (6) accession numbers of all database sequences with the best match, and (7) matching amino acid fragment sequence.

Cool?

Now its time to add taxon names so that the anvi’o parser can add the taxonomy to the contigs database. The addTaxonNames script needs location of the nodes.dmp file, location of the names.dmp file, the output from the Kaiju classification step, and an output name. Most importantly you must specify the taxonomic ranks to be included in the output as follows using the -r flag. Comma-separated, NO SPACES.

-r superkingdom,phylum,order,class,family,genus,species

So the full command will look something like this:

addTaxonNames -t /path/to/nodes.dmp \
              -n /path/to/names.dmp \
              -i gene_calls_nr.out \
              -o gene_calls_nr.names \
              -r superkingdom,phylum,order,class,family,genus,species

Whew. Now you are ready to run the anvi’o parser for Kaiju.

At this point its not a bad idea to make a copy of your contigs database –just in case. In order to get the Kaiju taxonomic profile into your contigs database you will run the following command with the output file from the addTaxonNames step:

anvi-import-taxonomy-for-genes -i gene_calls_nr.names \
                               -c contigs.db \
                               -p kaiju

After this, anvi’o will immediately throw the following error –not because anvi’o is mad at you, but because anvi’o is concerned about your well-being and doesn’t want you to ruin a perfectly good contigs database. anvi’o wants to make certain you are giving it a properly formatted input file:

Config Error: Anvi'o assumes you used this exact parameter during your kaiju run: '-r        
              superkingdom,phylum,order,class,family,genus,species'. If you haven't, you will
              run into trouble later. If you are positive that you did include that parameter
              to your run, re-run this program with `--just-do-it` flag

When you re-run the same command by including the --just-do-it flag, anvi’o will present you with a message indicating that it scanned your input file and gives you a second chance to back out of the arrangement.

If the phylum level names anvi’o presents don’t look correct, you can kill the who thing with CTRL+C –otherwise, the taxonomy will be added to your contigs database.

Thats it!

Now gene-level taxonomy from Kaiju should be available to you whenever it is appropriate in interactive anvi’o interfaces, and summary outputs.

Centrifuge

Anvi’o also has a parser for centrifuge. Please see this recipe to download and set up centrifuge.

Assuming you generated an anvi’o contigs database. To import taxonomy into this contigs database, first you will export all gene calls:

$ anvi-get-sequences-for-gene-calls -c CONTIGS.db -o gene-calls.fa

Then you will run the following command:

$ centrifuge -f -x $CENTRIFUGE_BASE/p+h+v/p+h+v gene-calls.fa -S centrifuge_hits.tsv

If the environment variable $CENTRIFUGE_BASE is not properly set, you will get an error. See the export instructions here to try again.

This step takes about one minute on my laptop for 40,000 genes.

When centrifuge is done running, you should find two files in your work directory, which you will import into anvi’o. These files are centrifuge_report.tsv, and centrifuge_hits.tsv. Just to make sure that they are not empty, feel free to run this command:

$ wc -l centrifuge_report.tsv centrifuge_hits.tsv
    4666 centrifuge_report.tsv
  215625 centrifuge_hits.tsv
  220291 total

Fine. It is time to import these results! To do this, you will use the program anvi-import-taxonomy with the parser for centrifuge:

$ anvi-import-taxonomy -c CONTIGS.db -i centrifuge_report.tsv centrifuge_hits.tsv -p centrifuge

You can use any file name you like, however, the order of input files is important: following the parameter -i, you should first declare the report file, and then the hits file.

This is it. If everything went alright, the interactive interface and anvi’o summary results should contain taxonomy information.

Simple matrix

If you have taxonomy information for your genes, but none of the parsers listed here helps you to import them, the following is the simplest way to get the taxonomical annotation of genes into a contigs database.

Basically first you can create The TAB-delimited input matrix that follows this format:

gene_callers_id t_domain t_phylum t_class t_order t_family t_genus t_species
1             Bacteroides fragilis
2             Bacteroides fragilis
3             Bifidobacterium longum
5             Bifidobacterium longum
7             Bifidobacterium longum
(…) (…) (…) (…) (…) (…) (…) (…)

And then you can use the following command to import it using the parser default_matrix:

$ anvi-import-taxonomy -c CONTIGS.db -i input_matrix.txt -p default_matrix

Not every gene call has to be in the matrix, and not every level of taxonomy has to be present, anvi’o will find a way to deal with that, but the more the merrier.