Added optional chapter 3

Author: rknx

_optionals/03-extra_tools.md

@@ -1,14 +1,90 @@
 ---
-title: "More bioinformatic tools"
-teaching: 
-exercises: 
-questions:
-- dsfgdf
+title: "Another bioinformatic tool: PyANI" 
 objectives:
-- dfgdf
-keypoints:
-- dfgdf
+- To calculated average nucleotide identity of a genome with other related genomes. 
 
 ---
 
-WIP
+In the regular lessons, we implemented three bioinformatic tools: 
+`blast` for homology search,
+`maaft` for sequence alignment, and
+`raxml` for phylogenetic analysis.
+
+In this section, we will discuss two more tools.
+
+## PyANI
+PyANI is an open-source python-based tool for calculating 
+Average Nucleotide Identity (ANI) between two or more sequences.
+When comparing two genomes, first syntenic regions are identified
+using tools such as `mummer` or `blast`.
+Then the nucleotide identity is calculated in the syntenic regions.
+
+The source code for **PyANI** is available at 
+[widdowquinn/pyani](https://github.com/widdowquinn/pyani){: target="_blank"}.
+The documentation for basic usage is available 
+[here](https://github.com/widdowquinn/pyani/blob/master/README_v_0_2_x.md){: target="_blank"}.
+
+**PyANI** v2 is available in Hipergator, but has to be loaded.
+The dependencies, `mummer` and `blast+` will be loaded together with `pyani`.
+
+~~~
+$ ml pyani
+~~~
+{: .language-bash}
+
+~~~
+Lmod is automatically replacing "python/3.8" with "pyani/0.2.10".
+~~~
+{: .output}
+
+We will be using the genomes present in `files/ani` for computing ANI.
+The file `UXhortspp.fasta` contains genome of a unknown *X. hortorum* species.
+The other sequences are genome of some *X. hortorum* pathovars 
+downloaded from NCBI.
+
+> ## Getting genome sequences from NCBI
+> PyANI has a script called `genbank_get_genomes_by_taxon.py` to download 
+> all genomes for a taxon from NCBI.
+> For usage, check the documentation linked above.
+{: .tips}
+
+The objective now is to perform pairwise comparisons of all reference genomes
+and calculate ANI. This can be performed with following command.
+
+~~~
+average_nucleotide_identity.py -i files/ani -o ani -m ANIm -g --gformat png,pdf
+~~~
+{: .language-bash}
+
+> - `average_nucleotide_identity.py` is the name of the script
+> - `-i` is used to specify directory containing input genomes/sequences.
+> - `-o` is used to specify output directory.
+> Note that the program will exit if this directory preexists.
+> - `-m` is used to specify mode for alignment of syntenic region. 
+> `ANIm` specifies `mummer` and `ANIb` specifies `blast+`.
+> - `-g` is used to generate graphic output, i.e., heatmap.
+> `--gformat` specifies the graphic output formats.
+{: .notes}
+
+~~~
+$ ls ani
+~~~
+{: .language-bash}
+
+~~~
+ANIm_alignment_coverage.pdf  ANIm_hadamard.pdf             ANIm_similarity_errors.pdf
+ANIm_alignment_coverage.png  ANIm_hadamard.png             ANIm_similarity_errors.png
+ANIm_alignment_coverage.tab  ANIm_hadamard.tab             ANIm_similarity_errors.tab
+ANIm_alignment_lengths.pdf   ANIm_percentage_identity.pdf  nucmer_output.tar.gz
+ANIm_alignment_lengths.png   ANIm_percentage_identity.png
+ANIm_alignment_lengths.tab   ANIm_percentage_identity.tab
+~~~
+{: .output}
+
+You can now transfer `ANIm_percentage_identity.png` 
+to your computer to view the heatmap.
+For numeric values, you can use `ANIm_percentage_identity.tab` table.
+
+<img src="/fig/ANIm_percentage_identity.png" height="500px">
+
+Based on ANI, the unknown strain seems to be *X. hortorum pv. gardneri*.