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Then you can provide FASTA files and get out sequence types (ST) for a given MLST database.
Two spreadsheets are outputted, one contains the allele number for each locus, and the ST (or nearest ST), the other contains the genomic sequence for each allele.  
If more than 1 allele gives 100% identity for a locus, the contaminated flag is set.
Optionally you can output a concatenated sequence in FASTA format, which you can then use with tree building programs.
New, unseen alleles are saved in FASTA format, with 1 per file, for submission to back to MLST databases.


# Citation
```"Multilocus sequence typing by blast from de novo assemblies against PubMLST", Andrew J. Page, Ben Taylor, Jacqueline A. Keane, The Journal of Open Source Software, (2016). doi: http://dx.doi.org/10.21105/joss.00118```

# Usage
The MLST databases must be downloaded first. This is something you would only do every now and again. You need to set the $MLST_DATABASES environment variable first to a location where you want to save your databases. If you use Docker, you can skip ...
```
Usage: download_mlst_databases [options]
   -c STR Config file containing details of MLST databases from pubMLST
   -b STR Directory where MLST databases are stored [$MLST_DATABASES]
   -h     Print this message and exit
   -v     Print version number and exit
```

The get_sequence_type script allows you to calculate the ST of a FASTA file against one or more database. If you dont provide the '-s' option, then every database will be searched.  If you wish to build a phylogenetic tree of then use the '-c' or '-y...
```
Usage: The get_sequence_type [options] *.fasta

   -s STR Species of MLST database (0 or more comma separated)
   -d INT Number of threads [1]
   -c     Output a FASTA file of concatenated alleles and unknown sequences 
   -y     Output a phylip file of concatenated alleles and unknown sequences
   -o STR Output directory [.]
   -a     Print out all available MLST databases and exit
   -h     Print this message and exit
   -v     Print version number and exit
```

# Input format
The input files must be in [FASTA format](https://en.wikipedia.org/wiki/FASTA_format) and contain nucleotide sequences. These can be full genome sequences, fragmented _de novo_ assemblies or individual genes. If the gene is truncated or split over 2 ...

# Outputs

## mlst_results.allele.csv
This is a tab separated spreadsheet containing the ST number of each input FASTA file and the corresponding allele numbers for each gene in the scheme. If one of the alleles is not contained in the database, then it will be flagged with 'U' and the 3...

Isolate | ST  |"New ST" |Contamination     | aroC | dnaN | hemD | hisD | purE | sucA | thrA
------- | --- | --------|------------------|------|------|------|------|------|------|-----
sample1 | ~559| Unknown |                  | 130  | 97   | 25   | 125  | U    | 9    | 101
sample2 | 518 |         |                  | 101  | 41   | 40   | 184  | 76   | 90   | 3
sample3 | 150 |         | purE-422,purE-84 | 130  | 97   | 25   | 125  | 422  | 9    | 101
sample4 | ~150| Novel   |                  | 130  | 95   | 25   | 125  | 422  | 9    | 101
sample5 |     |	Unknown |                  | U    | U    | U    | U    | U    | U    | U 

## mlst_results.genomic.csv
This spreadsheet is similar to the mlst_results.allele.csv spreadsheet, however it gives the full sequences of each allele instead of the allele number.

## *unknown.fa
You can choose to output any new alleles (-c) which are not contained in the MLST database. These can then be used to feedback to the curators maintaining the MLST databases, where they can be assigned allele numbers and profiles.

## concatenated_alleles.fa and concatenated_alleles.phylip
You can choose to output a multiple FASTA/Phylip alignment of all of the MLST genes concatenated together, where each sample is represented by a single sequence. This file can then be used as input to a phylogenetic tree building application (such as...

# Method
The user can decide to use a specific MLST scheme or search all of them. The first step is to generate a blastn database using makeblastdb from the alleles.  The input sequences are then blasted against the database using blastn.  If there is a 100% ...

#Installation
Instructions are given for installing the software via Docker (can be run on all operating systems),for Debian/Ubuntu distributions and HomeBrew/LinuxBrew.

##Docker
We have a docker container which is setup and ready to go. It includes a snapshot of the MLST databases from the day it was built.  To install it:

```
docker pull sangerpathogens/mlst_check
```

We have included some example data in the container, which can be run using this command:
```
docker run --rm -it -v /home/ubuntu/data:/data sangerpathogens/mlst_check get_sequence_type -s 'Salmonella enterica' /example/sample1.fa /example/sample2.fa /example/sample3.fa
```
Your results will then be in the /home/ubuntu/data directory (or whatever you have called it). 


To use the command with your own data place your FASTA files in /home/ubuntu/data (or substituting in your directories):
```
docker run --rm -it -v /home/ubuntu/data:/data sangerpathogens/mlst_check get_sequence_type -s 'Salmonella enterica' my_sample.fa
```
Your results will then be in the /home/ubuntu/data directory as previous. 

##Debian/Ubuntu
If you run Debian or Ubuntu it should be straightforward to install the software. These instructions assume you have root access. Run:

```
apt-get update -qq
apt-get install -y ncbi-blast+ cpanminus gcc autoconf make libxml2-dev zlib1g zlib1g-dev libmodule-install-perl
cpanm -f Bio::MLST::Check
```

Then you need to set a directory where you would like to store the MLST databases.
```
export MLST_DATABASES=/path/to/where_you_want_to_store_the_databases
```

Download the latest copy of the databases (run it once per month)
```
download_mlst_databases
```
Now you can use the script. For example,find the sequence types for all fasta files in your current directory:
```
get_sequence_type -s "Clostridium difficile" *.fa
```

##HomeBrew/LinuxBrew
If you run OSX, a non-Debian Linux or you do not have root access on your machine, you can use HomeBrew/LinuxBrew to install the dependancies.  First of all install [Homebrew](http://brew.sh/) (OSX) or [LinuxBrew](http://linuxbrew.sh/) (Linux).

```
brew tap homebrew/science
brew install cpanminus blast
```

Assuming you have setup perl modules to install in your local directory (~/perl5 in this case), this command will install this software and all its Perl dependancies:
```
cpanm --local-lib=~/perl5 -f Bio::MLST::Check
```
The process from this point is the same as installing with Debian.