--- # System prepended metadata title: FastANI example tags: [General] --- --- tags: General title: FastANI example --- # FastANI example [FastANI](https://github.com/ParBLiSS/FastANI#fastani) is tool for generating a metric telling us something about how similar two genomes are called average nucleotide identity (ANI). It can be run on many genomes, but each value tells us something about a pairwise comparison between two genomes. It is on a scale between 1 and 100 and it is (roughly) the percent of nucleotide bases that are identical between the two genomes 👍 [toc] ## Conda install Using [mamba](https://astrobiomike.github.io/unix/conda-intro#bonus-mamba-no-5) on top of conda, 'cause it's faster. Including my [bit](https://github.com/AstrobioMike/bit#readme) package to be able to download example genomes quickly. ```bash mamba create -n fastani -c conda-forge -c bioconda -c defaults -c astrobiomike fastani bit -y conda activate fastani ``` ## Getting example genomes The input genomes need to be fasta files, so here just making a list with a few accessions to pass to `bit-dl-ncbi-assemblies`. Using these: | isolate ID | tax | assembly acc | isolation date | isolation location | |--|--|--|--|--| | s1 | S. epidermidis | GCA_016809475.1 | 8/14/06 | Node 1 | | s2 | S. epidermidis | GCA_016808675.1 | 8/14/06 | Node 1 | | s3 | S. epidermidis | GCA_016809435.1 | 8/14/06 | U.S. LAB | | s4 | S. epidermidis | GCA_016808655.1 | 7/15/09 | Node 1 | | s49 | S. epidermidis | GCA_016808775.1 | 4/16/15 | Node 2 | | s53 | S. epidermidis | GCA_016808695.1 | 11/16/15 | PMM | ```bash printf "GCA_016809475.1\nGCA_016808675.1\nGCA_016809435.1\nGCA_016808655.1\nGCA_016808775.1\nGCA_016808695.1\n" > target-accs.txt bit-dl-ncbi-assemblies -f fasta -w target-accs.txt -j 3 ``` ## Example running FastANI to compare all We just need a file holding the paths to the fasta files we want to compare, which right now, this will work to create that: ```bash ls *.gz > genome-paths.txt head genome-paths.txt | sed 's/^/# /' # GCA_016808655.1.fa.gz # GCA_016808675.1.fa.gz # GCA_016808695.1.fa.gz # GCA_016808775.1.fa.gz # GCA_016809435.1.fa.gz # GCA_016809475.1.fa.gz ``` And running fastANI: ```bash fastANI --ql genome-paths.txt --rl genome-paths.txt -t 4 -o fastANI-out-raw.tsv ``` The output looks like this, with the columns: 1. query 2. reference 3. ANI value 4. number of fragments mapped 5. total number of query fragments ```bash cat fastANI-out-raw.tsv | sed 's/^/# /' # GCA_016808655.1.fa.gz GCA_016808655.1.fa.gz 100 793 795 # GCA_016808655.1.fa.gz GCA_016808695.1.fa.gz 99.6563 762 795 # GCA_016808655.1.fa.gz GCA_016808775.1.fa.gz 99.388 736 795 # GCA_016808655.1.fa.gz GCA_016809435.1.fa.gz 99.377 752 795 # GCA_016808655.1.fa.gz GCA_016809475.1.fa.gz 96.8913 737 795 # GCA_016808655.1.fa.gz GCA_016808675.1.fa.gz 96.756 734 795 # GCA_016808675.1.fa.gz GCA_016808675.1.fa.gz 100 780 786 # GCA_016808675.1.fa.gz GCA_016809475.1.fa.gz 98.5649 734 786 # GCA_016808675.1.fa.gz GCA_016808775.1.fa.gz 96.8927 727 786 # GCA_016808675.1.fa.gz GCA_016808655.1.fa.gz 96.7925 730 786 # GCA_016808675.1.fa.gz GCA_016809435.1.fa.gz 96.7467 736 786 # GCA_016808675.1.fa.gz GCA_016808695.1.fa.gz 96.7231 739 786 # GCA_016808695.1.fa.gz GCA_016808695.1.fa.gz 100 793 795 # GCA_016808695.1.fa.gz GCA_016808655.1.fa.gz 99.684 762 795 # GCA_016808695.1.fa.gz GCA_016808775.1.fa.gz 99.4361 743 795 # GCA_016808695.1.fa.gz GCA_016809435.1.fa.gz 99.372 745 795 # GCA_016808695.1.fa.gz GCA_016809475.1.fa.gz 96.9338 742 795 # GCA_016808695.1.fa.gz GCA_016808675.1.fa.gz 96.68 740 795 # GCA_016808775.1.fa.gz GCA_016808775.1.fa.gz 100 776 779 # GCA_016808775.1.fa.gz GCA_016809435.1.fa.gz 99.4809 738 779 # GCA_016808775.1.fa.gz GCA_016808695.1.fa.gz 99.4482 743 779 # GCA_016808775.1.fa.gz GCA_016808655.1.fa.gz 99.4255 736 779 # GCA_016808775.1.fa.gz GCA_016809475.1.fa.gz 96.963 736 779 # GCA_016808775.1.fa.gz GCA_016808675.1.fa.gz 96.8906 727 779 # GCA_016809435.1.fa.gz GCA_016809435.1.fa.gz 100 808 811 # GCA_016809435.1.fa.gz GCA_016808775.1.fa.gz 99.4869 740 811 # GCA_016809435.1.fa.gz GCA_016808655.1.fa.gz 99.3886 757 811 # GCA_016809435.1.fa.gz GCA_016808695.1.fa.gz 99.3089 753 811 # GCA_016809435.1.fa.gz GCA_016809475.1.fa.gz 96.9509 727 811 # GCA_016809435.1.fa.gz GCA_016808675.1.fa.gz 96.8241 728 811 # GCA_016809475.1.fa.gz GCA_016809475.1.fa.gz 100 809 810 # GCA_016809475.1.fa.gz GCA_016808675.1.fa.gz 98.6019 735 810 # GCA_016809475.1.fa.gz GCA_016809435.1.fa.gz 97.0084 724 810 # GCA_016809475.1.fa.gz GCA_016808695.1.fa.gz 96.9251 735 810 # GCA_016809475.1.fa.gz GCA_016808655.1.fa.gz 96.9205 728 810 # GCA_016809475.1.fa.gz GCA_016808775.1.fa.gz 96.9141 735 810 ``` ### Note on output There are self-comparisons in the default output. And as with all comparisons like these, A vs B can be slightly different than B vs A (both are in the outputs). I have some R code to create a new table always use the longer genome as the query, proportion aligned is reported consistently as being out of the longer of the two genomes. Let me know if you'd like it! ## BONUS: Some clean-up I do I like to remove the ones that are comparing to themselves (same query and reference genome), and I like to add a column with percent aligned, which is useful information. Can be done any number of ways, here is doing these two things with awk, then sorting by percent aligned and then percent identity: ```bash awk '$1 != $2 ' fastANI-out-raw.tsv | awk ' { palign = $4 / $5 * 100 } { print $0"\t"palign } ' | sort -nr -k 6,6 -k 4,4 > fastANI-out.tsv ``` Now there are no rows with self-contrasts, and there is a new column at the end of percent aligned: ```bash cat fastANI-out.tsv | sed 's/^/# /' # GCA_016808695.1.fa.gz GCA_016808655.1.fa.gz 99.684 762 795 95.8491 # GCA_016808655.1.fa.gz GCA_016808695.1.fa.gz 99.6563 762 795 95.8491 # GCA_016808775.1.fa.gz GCA_016808695.1.fa.gz 99.4482 743 779 95.3787 # GCA_016808775.1.fa.gz GCA_016809435.1.fa.gz 99.4809 738 779 94.7368 # GCA_016808655.1.fa.gz GCA_016809435.1.fa.gz 99.377 752 795 94.5912 # GCA_016808775.1.fa.gz GCA_016809475.1.fa.gz 96.963 736 779 94.4801 # GCA_016808775.1.fa.gz GCA_016808655.1.fa.gz 99.4255 736 779 94.4801 # GCA_016808675.1.fa.gz GCA_016808695.1.fa.gz 96.7231 739 786 94.0204 # GCA_016808695.1.fa.gz GCA_016809435.1.fa.gz 99.372 745 795 93.7107 # GCA_016808675.1.fa.gz GCA_016809435.1.fa.gz 96.7467 736 786 93.6387 # GCA_016808695.1.fa.gz GCA_016808775.1.fa.gz 99.4361 743 795 93.4591 # GCA_016808675.1.fa.gz GCA_016809475.1.fa.gz 98.5649 734 786 93.3842 # GCA_016809435.1.fa.gz GCA_016808655.1.fa.gz 99.3886 757 811 93.3416 # GCA_016808695.1.fa.gz GCA_016809475.1.fa.gz 96.9338 742 795 93.3333 # GCA_016808775.1.fa.gz GCA_016808675.1.fa.gz 96.8906 727 779 93.3248 # GCA_016808695.1.fa.gz GCA_016808675.1.fa.gz 96.68 740 795 93.0818 # GCA_016808675.1.fa.gz GCA_016808655.1.fa.gz 96.7925 730 786 92.8753 # GCA_016809435.1.fa.gz GCA_016808695.1.fa.gz 99.3089 753 811 92.8483 # GCA_016808655.1.fa.gz GCA_016809475.1.fa.gz 96.8913 737 795 92.7044 # GCA_016808655.1.fa.gz GCA_016808775.1.fa.gz 99.388 736 795 92.5786 # GCA_016808675.1.fa.gz GCA_016808775.1.fa.gz 96.8927 727 786 92.4936 # GCA_016808655.1.fa.gz GCA_016808675.1.fa.gz 96.756 734 795 92.327 # GCA_016809435.1.fa.gz GCA_016808775.1.fa.gz 99.4869 740 811 91.2454 # GCA_016809475.1.fa.gz GCA_016808775.1.fa.gz 96.9141 735 810 90.7407 # GCA_016809475.1.fa.gz GCA_016808695.1.fa.gz 96.9251 735 810 90.7407 # GCA_016809475.1.fa.gz GCA_016808675.1.fa.gz 98.6019 735 810 90.7407 # GCA_016809475.1.fa.gz GCA_016808655.1.fa.gz 96.9205 728 810 89.8765 # GCA_016809435.1.fa.gz GCA_016808675.1.fa.gz 96.8241 728 811 89.7657 # GCA_016809435.1.fa.gz GCA_016809475.1.fa.gz 96.9509 727 811 89.6424 # GCA_016809475.1.fa.gz GCA_016809435.1.fa.gz 97.0084 724 810 89.3827 ``` And if wanting a header: ```bash cat <( printf "query\treference\tANI\tnum_frags_mapped\ttotal_query_frags\tpercent_aligned\n" ) fastANI-out.tsv > fastANI-out-with-header.tsv ``` ```bash column -ts $'\t' fastANI-out-with-header.tsv | head | sed 's/^/# /' # query reference ANI num_frags_mapped total_query_frags percent_aligned # GCA_016808695.1.fa.gz GCA_016808655.1.fa.gz 99.684 762 795 95.8491 # GCA_016808655.1.fa.gz GCA_016808695.1.fa.gz 99.6563 762 795 95.8491 # GCA_016808775.1.fa.gz GCA_016808695.1.fa.gz 99.4482 743 779 95.3787 # GCA_016808775.1.fa.gz GCA_016809435.1.fa.gz 99.4809 738 779 94.7368 # GCA_016808655.1.fa.gz GCA_016809435.1.fa.gz 99.377 752 795 94.5912 # GCA_016808775.1.fa.gz GCA_016809475.1.fa.gz 96.963 736 779 94.4801 # GCA_016808775.1.fa.gz GCA_016808655.1.fa.gz 99.4255 736 779 94.4801 # GCA_016808675.1.fa.gz GCA_016808695.1.fa.gz 96.7231 739 786 94.0204 # GCA_016808695.1.fa.gz GCA_016809435.1.fa.gz 99.372 745 795 93.7107 ``` --- **Side notes** > It looks like there are some stable Staph populations persisting there across time. The highest ANI was between s53 (11/16/15 from PMM) and s4 (7/15/09 Node 1): 99.7% ANI with 95% aligning The lowest ANI was between s3 (8/14/06 from U.S.LAB) and s1 (8/14/06 Node 1). s1 to s2 (same date and location): 98.6% ANI with 90.1% aligning (or 98.6% ANI with 93.4% aligning) ---