rastair provides a set of tools to process TAPS sequencing data.

Installation

rastair is written in Rust and can be built using cargo or docker.

Cargo

First, ensure that the CLI tool cargo is installed. See instructions to install. Then run the following command to install rastair:

cargo install --git https://bitbucket.org/bsblabludwig/rastair.git

Run the following command to verify your installation:

rastair --help

We are planning to release statically compiled binaries for different systems in the future.

Docker

A Dockerfile is provided in the repository. To build the docker image, run:

git clone https://bitbucket.org/bsblabludwig/rastair.git
cd rastair
VERSION=$(git describe)
docker build -t rastair:$VERSION .

rastair can now be run in a docker container. For example, to call methylation from a bam file, run:

# Assuming you have a bam file in /local/path/to/data/my.bam and a fasta file in /local/path/to/data/my.fasta, they can be volume-mounted into the container with the -v flag to be processed by rastair
docker run -v /local/path/to/data:/data rastair:$VERSION rastair call --fasta-file /data/my.fasta /data/my.bam

Available commands

rastair offers a number of sub-commands, each with their own set of options. You can get a list of available commands by typing

rastair --help

Currently, the following sub-commands are available:

Fast and flexible extraction of methylation information from bam files

Usage: rastair [OPTIONS] [COMMAND]

Commands:
  call      Call methylation at CpG positions from a bam file
  per-read  Call methylation per read. This will produce a bed file that list the methylation status of all CpGs in every read that overlaps a CpG, plus some other metadata
  mbias     Calculate conversion per base position in read
  map-cpgs  print a map of all CpGs in a fasta file and their indices as a bed file
  bed2pat   Utility function to convert per-read bed files to PAT files compatible with wgbstools and UXMtools
  help      Print this message or the help of the given subcommand(s)

Options:
  -v, --verbosity...
  -h, --help          Print help
  -V, --version       Print version

In general, you can use --help on all rastair sub-commands to get detailed instructions on the available options.

Utility scripts

rastair is designed to process (relatively) raw files into a format that is useful for downstream analysis. Any plotting and summarising is meant to happen downstream of rastair. We provide a number of convenience R scripts in the scripts directory. Currently, these include:

• plot_mbias.R : This takes the output of rastair mbias and generates a figure of OT/OB conversion per read position. It also calculates suggested cut-off parameters for --nOT and --nOB to use in rastair call. This script optionally depends on the ggplot2 library for making figures
• calculate_conversion.R : Generate a summary statistic for the conversion rate in a genomic region

Notes

Soft-clipping option syntax

The correction of "m-bias", ie the loss of conversion around read ends, is an important aspect of several rastair sub-commands (e.g. call, bed2pat). The command-line argument for this was inspired by MethylDackel. However, we decided to simplify the argument and make it more consistent (in our perspective, at least). This means we only have one set of parameters --nOT and --nOB. Each of them takes a comma-separated list of 4 integers, denoting the number of bases from the start/end of read 1/2 that should be ignored. However, unlike MethylDackel, rastair accounts for read orientation, so the command like arguments always refer to the physical start/end position of the read, not the position in the alignment. To give an example: imagine the following read pair

    000000000111111111122
    123456789012345678901
R1: CG--------TG--------->
R2:                         <AC-------------GC-
                             876543210987654321
                             111111111000000000

This "F1R2" read represents the OT (ie R1 is the OT, and R2 is the reverse complement of the OT). A parameter of --nOT 0,5,0,5 will exclude the A at position 18 in R2, because it ocurrs within 5 bases from the end of R2 in read coordinates, not in reference coordinates.

Performance considerations

Most commands in rastair can be run in multi-threaded mode using the -@ <ncores> parameter. However, the performance increase has diminishing returns, as the threads have to eventually synchronise for writing to the output. We have found that increasing the number of BAM read threads to 2 (--read-threads 2) in combination with 4 to 6 processing threads (-@ 4) seems to perform best on a high-io-throughput system.

License

This software is made available under the terms of the GNU Affero General Public License v3. If you require a more restrictive license for commercial purposes, please contact the authors to discuss alternative arrangements.

THE SOFTWARE IS PROVIDED "AS IS", WITHOUT WARRANTY OF ANY KIND, EXPRESS OR IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, TITLE AND NON-INFRINGEMENT. IN NO EVENT SHALL THE COPYRIGHT HOLDERS OR ANYONE DISTRIBUTING THE SOFTWARE BE LIABLE FOR ANY DAMAGES OR OTHER LIABILITY, WHETHER IN CONTRACT, TORT OR OTHERWISE, ARISING FROM, OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN THE SOFTWARE.