Pipeline to perform in a single step mapping-by-sequencing and mutation identification. ##General description This pipeline has been originally developped for the nematode species Oscheius tipulae by the Félix lab. This repository contains all data to perform mapping-by-sequencing with Oscheius tipulae using the reference strain CEW1 and the mapping strain JU170 (see Félix Lab Strain database). However, this is a versatile pipeline that allows working with any species and any mapping strains (Read below "How to adapt it to my purpose ?").
Andalusian_Mapping is mainly encoded in bash and it can be run on Linux (tested on Ubuntu 16.04 LTS) and MacOS (not Windows). It uses popular third-party NGS softwares (six exactly, see "Requirements") that must be installed in the computing machine.
Many bio-informatic steps are needed from the raw sequencing data to the final identification of candidate mutations. Andalusian_Mapping allows pipelining them all via one simple command line. This pipeline can then be used routinely with very basic knowledge in shell coding.
Recently, Cloudmap, a nice automatic pipeline of mapping-by-sequencing/mutant-identification has been developped and integrated to Galaxy, with an intuitive and user-friendly GUI. It works very well with model species like Caenorhabditis elegans or Arabidopsis thaliana. However, all genomes are not available in Galaxy. We had to develop Anadalousian-Mapping to work with the non-model species Oscheius tipulae, which had no published genome. But we tried to keep simple procedures so that a large audience of biologists (and not only bio-informaticians) could find it useful.
We propose that Andalusian-Mapping will be an attractive alternative to other pipelines for people who need to work with non-reference genome assembly but with minimal efforts in computing/bio-informatics. Once the set-up is done, the procedure is really simple: one command line only.
Here are some advantages of Andalusian-Mapping that may match your needs:
- Most third-party software easy to install and so popular that they might already be on your system (e.g. R, bwa, samtools) ;
- The most complicated software to install are GATK and snpEff but they both have very clear tutorials and debug forum online. If it remains a problem for you, any people with intermediate computing skills should easily and quickly debug this step for you ;
- Once the set-up is done, the procedure is really simple: one command line only ;
- You can run it locally and don't need to queue your job in a distant server ;
- You can adapt it to any reference genomes. Hence, you can study non-model organisms or use an alternative assembly of a model-organism (different strain depedning on the design of your mapping cross).
- You manage the versions of your software: you can either benefit from the latest improvements of third-party software or freeze you set-up in the configuration you prefer.
Andalusian-Mapping takes as input paired-end reads from a bulk sequencing of a F2 population generated through a cross of a (ideally recessive) mutant strain with a mapping strain.
- It outputs plots of allele frequency (mutant strain allele / mapping-strain allele) for each scaffolds/Chromosome present in the reference genome. This allows to visualy map the region linked to the mutation selected in the F2 population.
- It outputs an annotated vcf file containing the mutations linked to the phenotype-causing mutations with their functional impacts
- It outputs a text file that select the best candidate mutations based on the predicted functional impact.
- bwa version 0.7.5a-r405 or later
- samtools version 0.1.18 or later
- Picard Version 1.110 or later
- GATK Version 3.7 or later
- R Version 3.3 or later, with ggplot2 package.
- snpEff Version 4.1g or later
- Paired-end sequencing data of a bulk F2 mapping population.
- The reference genome of your organism
- a list of high confidence mapping SNPs (or other variants) between the mapping strain and the background strain and the respective vcf files of these two strains.
Run a simple and stereotyped command line:
bash (/path/to/)andalousian-map_Portable.sh (/path/to/)Config-file_mysample.txt
andalousian-map_Portable.sh is a bash script (no need to edit).
For each new analysis, you only change the configuration file ('Config-file_mysample.txt' in the command above), which is a simple text file. It only contains paths to data or scripts and the name (prefix) you want to give to your output files
A template and an example configuration files are given in this folder.
Ploting and filtering steps can be run separately with the two scripts map_mutant_Portable.sh and find_gene_Portable.sh.
This repository contain all data to perform mapping-by-sequencing/mutant-identification in Oscheius tipulae with the two strains CEW1 and JU170.
If you want to use this pipeline with another organism, you first need a reference genome (a draft, fragmented assembly should work). Then you need to sequence the backgroud strain and the mapping strain and perform a variant calling to define a list of mapping SNPs.
We named our pipeline "Andalusian_Mapping" in reference to the "Hawaïan Mapping" procedure developped in C. elegans, involving the Hawaïan mapping strain CB4856. Our O. tipuale mapping strain is an Andalusian wild isolate sampled in Sevilla, Spain (see the Félix lab Strain database).