ReferenceAlignmentGenerator

This program uses SMALT to align reads to the reference genome, and then uses SAMtools to generate a consensus genome for each read file. The alignments of genes are extracted, alignments with gaps removed, and alignments for core and accessory genes are outputted. There are several additional features that have been added, as described in "Usage".

Installation

For basic installation (making reference-guided alignments), clone this github repository to your workstation and install the program RefAligner via pip:

• pip install ~/go/src/github.com/kussell-lab/ReferenceAlignmentGenerator/RefAlignGenerate

The following dependencies need to be installed:

• SMALT
• SAMtools
• Parallel
• sra-tools

As well as several in-house developed programs found in this repo:

• go get -u github.com/kussell-lab/ReferenceAlignmentGenerator/GenomicConsensus
• go get -u github.com/kussell-lab/ReferenceAlignmentGenerator/FilterGaps
• go get -u github.com/kussell-lab/ReferenceAlignmentGenerator/CollectGeneAlignments
• go get -u github.com/kussell-lab/ReferenceAlignmentGenerator/SplitGenome

We have tested this on Mac OS 11.4 and on the NYU HPC (which is Linux-based) using Python 3.8 and 3.9, Go version 1.15.7 and 1.16, SMALT 0.7.6, SAMtools 1.11, sra-tools 2.10.9, parallel 20201022

Basic Usage

After installation, enter RefAligner --help into your terminal to see inputs. For an example, see the RUN_ME.sh file in the "hinfluenzae_example" subdirectory. For large jobs (>500 genomes), we recommend doing each of the steps that the RefAligner does separately, because there may be an issue with one step that creates downstream problems. See the following file for the step-by-step process:

~/go/src/github.com/kussell-lab/ReferenceAlignmentGenerator/RefAlignGenerate/RefAlignGenerate/RefAligner/cli.py

Additional Features

(1) To split XMFA files into XMFA files which only include genes which are in a certain percentage of strains, (e.g., 50-70% of strains) use the GeneBins program, which can be installed via:

• go get -u github.com/kussell-lab/ReferenceAlignmentGenerator/GeneBins

After installation, enter GeneBins --help into your terminal to see inputs/outputs.

(2) To measure the frequency of gaps in an XMFA file generated using the RefAligner, use MeasureGaps, which can be installed via:

• go get -u github.com/kussell-lab/ReferenceAlignmentGenerator/MeasureGaps

After installation, enter MeasureGaps --help into your terminal for inputs/outputs.

Example

For an example of how to run RefAligner, see the directory "hinfluenzae_example" in this repo. To run the example, run the RUN_ME.sh script. Downloads and read mapping are the time limiting step. To run the full example takes ~2-3 hours on a standard PC. To skip the download step and run everything else, use the flag --skipdownloads=True. This should then only take ~5 minutes to run. Example outputs are given in the folder as well.