Itol tutorial


  • Phylogenetic placement re-re-visited
  • Beginner Guide to Bacteriophage Genome Assembly 1. Introduction
  • For example, I have a table of resistance genes and mutations detected in each bacterial strain, and a phylogenetic tree showing the relationships between strains. I want to quickly and easily plot the tree and data together, so I can see whether the resistance genes are clustered together in a single clade or lineage, or if they are cropping up in lots of unrelated strains.

    There are quite a few tools out there that can do something like this, but they all seem to have their drawbacks and issues, so I ended up hacking up an R script to do what I want. Here is a quick round-up of the tools I found, and the script I came up with. You just upload a newick tree file and tables of data in various formats described here , and it can display all kinds of data see examples on the front page.

    Figures can be exported in PDF and other formats, which is great for publications. The data has to be the correct format for iTOL before uploading. Color-strip types are good for showing categorical variables, e.

    I often want to look at the distribution of different types of gyrA mutations that confer reduced resistance to fluoroquinolone-based drugs, and I need to have each mutation in a different colour. Note that this is a screenshot from my browser, as the export option was not functioning correctly today a drawback of web-based services. Some of this can be got around by editing the newick file first in some other program, and changing colours and weights later by adjusting the PDF output by iTOL.

    It is web-based. You can however establish a private account, and keep track of your trees in separate projects and groups, which is nice. There is also an API and batch access , if you want to link it in with other web services. This is essentially a package for navigating and displaying phylogenetic trees, and it has some really great features. It allows you to display sequence alignments, images, protein domains, heatmaps, graphs etc overlaid on tree nodes, leaves or next to the tree, and has the option of half-circle tree plots in addition to the usual full circle tree layout.

    For examples see the web page. ETE comes with its own graphical interface for displaying trees, as well as being able to write them out directly to image files. It can ladderized trees, but not when you are using the heatmap display. So unless your tree happens to be naturally ladderized, it can look a bit strange.

    Like iTOL, you just need a tree in newick format, and a matrix representing your heatmap. Unlike iTOL, you need to make sure first that the names in your tree and matrix match up At the end of the post are some example code for how to colour in the leaf nodes with location data, and add a colour strip indicating our resistance mutations, based on the ETE2 tutorial.

    I also had problems with the branch leading from the root of the tree being drawn as super long by ETE, which makes it awkward to render as a nice image. For heatmaps, you have virtually no control over the colour scheme. It can also facilitate building the tree drawing into other data analysis pipelines much like iTOL batch could be, I suppose.

    But for many people this will be a hindrance. It can contain a phylogenetic tree and data matrix, mainly for analysis rather than data display, although there are a couple of functions for plotting the data against the tree. Instead it represents values in the data matrix by the size of circles or squares laid out in a grid.

    Posted on May 13, by Jeff I use phylogenetic placement, namely the program pplacer , in a lot of my publications. It is also a core part of of the paprica metabolic inference pipeline. As a result I field a lot questions from people trying to integrate pplacer into their own workflows.

    Although the Matsen group has done an excellent job with documentation for pplacer , guppy , and taxtastic , the three programs you need to work with to do phylogenetic placement from start to finish see also EPA , there is still a steep learning curve for new users. In the hope of bringing the angle of that curve down a notch or two, and updating my previous posts on the subject here and here , here is a complete, start to finish example of phylogenetic placement, using 16S rRNA gene sequences corresponding to the new tree of life recently published by Hug et al.

    To follow along with the tutorial start by downloading the sequences here. You can use any number of alignment and tree building programs to create a reference tree for phylogenetic placement. After a lot of experimentation this combination seems to be produce the most correct topologies and best supported trees. You can find that at the Rfam database here. You may also need to swap out the model used by RAxML.

    The workflow will follow these steps: Create an alignment of the reference sequences with Infernal Create a phylogenetic tree of the alignment Create a reference package from the alignment, tree, and stats file Proceed with the phylogenetic placement of your query reads Create an alignment of the reference sequences The very first thing that you need to do is clean your sequence names of any wonky punctuation.

    This is something that trips up almost everyone. Now feed it to RAxML to build a tree. Depending on the size of the alignment this can take a little bit. You can do this manually, or you can have RAxML try to root the tree for you.

    The work around is to add the confidence scores to the already generated rooted tree, so that you a version of the tree with out without scores. You will feed the scored tree to Taxtastic with the stats file from the unscored tree we already generated.

    You can probably accept most of the flags for the previous commands as is. You will need to use guppy to convert this json-format file to information that is readable by human. The two most useful guppy commands in my experience for a basic look at your data are: Generate an easily parsed csv file of placements, with only a single placement reported for each query read. The problem with this is that esl-alimerge needs the original sto file produced by cmalign, and that file contains duplicate sequences not used to build the reference tree.

    Now feed it to RAxML to build a tree. Depending on the size of the alignment this can take a little bit. You can do this manually, or you can have RAxML try to root the tree for you.

    Phylogenetic placement re-re-visited

    The work around is to add the confidence scores to the already generated rooted tree, so that you a version of the tree with out without scores. You will feed the scored tree to Taxtastic with the stats file from the unscored tree we already generated. You can probably accept most of the flags for the previous commands as is.

    You will need to use guppy to convert this json-format file to information that is readable by human.

    Beginner Guide to Bacteriophage Genome Assembly 1. Introduction

    Code and examples are below. However I like to work with R and find it easier for many things, so I also wrote an R function to plot trees with data. This just uses the ape package for plotting the tree object, and other basic R functions for plotting data alongside the tree. I know there are loads more out there, and this is not meant to be an exhaustive list but my personal recommendations… but if you want to share your own favourites feel free to add a comment to this post.

    Annotating trees with data: Holt lab scripts We have two work-horse scripts for plotting trees with data, one based on R using ape and one based on Python using the ete2 package. Each can do slightly different things. The biggest difference is that the R script is restricted to rectangular trees and works best for plotting associated data as text columns and heatmaps, like this example taken from Holt et al, PNAS of a tree of Vietnamese Shigella sonnei, with tips coloured by city of isolation, and heatmap indicating the presence black or absence white of accessory genes.

    You can however establish a private account, and keep track of your trees in separate projects and groups, which is nice. There is also an API and batch accessif you want to link it in with other web services.

    This is essentially a package for navigating and displaying phylogenetic trees, and it has some really great features. It allows you to display sequence alignments, images, protein domains, heatmaps, graphs etc overlaid on tree nodes, leaves or next to the tree, and has the option of half-circle tree plots in addition to the usual full circle tree layout.

    For examples see the web page. ETE comes with its own graphical interface for displaying trees, as well as being able to write them out directly to image files. It can ladderized trees, but not when you are using the heatmap display. So unless your tree happens to be naturally ladderized, it can look a bit strange. Like iTOL, you just need a tree in newick format, and a matrix representing your heatmap.


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