FAME Metagenomics workshop 2022
The NCBI has a very nice tool for identifying Anti-Micriobial Resistance (AMR) genes, as well as virulence factors and toxins.
Installing AMRFinder is easily done with conda.
# create a new environemnt
conda create -n amrfinder
# activate the environment
conda activate amrfinder
# install AMRFinder (specify bioconda and conda-forge channels)
conda install -c bioconda -c conda-forge ncbi-amrfinderplus
Running AMRFinder is relatively easy. Before the first time you run AMRFinder, you need to download its databases.
amrfinder --update
This will download the database files for you. Now you can start looking for AMRs! If you have an assembly, or some sequencing reads, you would use the nucleotide option.
# run on an assembly
amrfinder -n assembly.fasta > amrfinder.output.tsv
# run on some reads
amrfinder -n someReads.fasta > amrfinder.output.tsv
# oh no, I have fastq.gz files, not fasta files
zcat reads.fastq.gz | sed -n '1~4s/^@/>/p;2~4p' > reads.fasta
amrfinder -n reads.fasta > amrfinder.output.tsv
If you’ve already annotated your genome and have a bunch of predicted protein sequences, you can feed these into amrfinder instead.
amrfinder -p proteins.fasta > amrfinder.output.tsv
If you have an annotated genome, you can feed in both the protein and nucleotide sequences. HOWEVER, amrfinder will want to know where the protein sequences belong on the genome. You feed it this information with a GFF file, which specifies the genome coordinates of all proteins. The problem you will probably encounter with GFF files in general is that they are often not formatted correctly, or different programs will want them formatted in slightly different ways. Assuming you have a GFF file that AMRFinder is happy with:
amrfinder -p proteins.fasta -n assembly.fasta -g annotations.gff > amrfinder.output.tsv
The output of AMRFinder is a tab separated file (TSV). Everything should be TSV. The output looks like this:
Protein identifier Contig id Start Stop Strand Gene symbol Sequence name Scope Element type Element subtype Class Subclass Method Target length Reference sequence length % Coverage of reference sequence % Identity to reference sequence Alignment length Accession of closest sequence Name of closest sequence HMM id HMM description
NA ERR1356692:1:261704 1 123 - dfrB5 trimethoprim-resistant dihydrofolate reductase DfrB5 core AMR AMR TRIMETHOPRIM TRIMETHOPRIM PARTIAL_CONTIG_ENDX 41 78 52.56 92.68 41 WP_063844482.1 trimethoprim-resistant dihydrofolate reductase DfrB5 NA NA
NA ERR1543789:16:20590 3 251 - lnu(C) lincosamide nucleotidyltransferase Lnu(C) core AMR AMR LINCOSAMIDE LINCOSAMIDE PARTIAL_CONTIG_ENDX 83 164 50.61 96.39 83 WP_063851341.1 lincosamide nucleotidyltransferase Lnu(C) NA NA
NA ERR1301981:5:3301 2 250 - lnu(C) lincosamide nucleotidyltransferase Lnu(C) core AMR AMR LINCOSAMIDE LINCOSAMIDE PARTIAL_CONTIG_ENDX 83 164 50.61 98.80 83 WP_063851341.1 lincosamide nucleotidyltransferase Lnu(C) NA NA
NA ERR1664649:16:12246 3 299 - erm(37) 23S rRNA (adenine(2058)-N(6))-methyltransferase Erm(37) core AMR AMR MACROLIDE MACROLIDE PARTIAL_CONTIG_ENDX 99 179 55.31 100.00 99 WP_003900446.1 23S rRNA (adenine(2058)-N(6))-methyltransferase Erm(37) NA NA
NA ERR1664649:21:8743 3 299 - erm(37) 23S rRNA (adenine(2058)-N(6))-methyltransferase Erm(37) core AMR AMR MACROLIDE MACROLIDE PARTIAL_CONTIG_ENDX 99 179 55.31 100.00 99 WP_003900446.1 23S rRNA (adenine(2058)-N(6))-methyltransferase Erm(37) NA NA
NA ERR1664649:25:49979 1 300 - aac(2')-Ic aminoglycoside N-acetyltransferase AAC(2')-Ic core AMR AMR AMINOGLYCOSIDE GENTAMICIN/TOBRAMCYIN PARTIAL_CONTIG_ENDX 100 181 55.25 100.00 100 WP_003899880.1 aminoglycoside N-acetyltransferase AAC(2')-Ic NA NA
NA ERR1356738:1:295758 8 124 - dfrB trimethoprim-resistant dihydrofolate reductase DfrB core AMR AMR TRIMETHOPRIM TRIMETHOPRIM PARTIAL_CONTIG_ENDX 39 78 50.00 94.87 39 WP_063844479.1 trimethoprim-resistant dihydrofolate reductase DfrB6 NA NA
NA ERR1467153:1:136128 13 243 - qacH quaternary ammonium compound efflux SMR transporter QacH core STRESS BIOCIDE QUATERNARY AMMONIUM QUATERNARY AMMONIUM PARTIAL_CONTIG_ENDX 77 125 61.60 89.61 77 WP_000121134.1 quaternary ammonium compound efflux SMR transporter QacH NA NA
NA ERR1467153:2:333502 2 214 + qacL quaternary ammonium compound efflux SMR transporter QacL core STRESS BIOCIDE QUATERNARY AMMONIUM QUATERNARY AMMONIUM PARTIAL_CONTIG_ENDX 71 110 64.55 97.18 71 WP_015060824.1 quaternary ammonium compound efflux SMR transporter QacL NA NA
As you can see this shows you what predicted AMR genes etc are identified in your samples, and importantly the sequence IDs and coordinates. You can look at the alignment metrics to help you decide if you believe the gene is there and in tact etc.