Zipped case data

Update Readme

README.md

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 # DBA
-DNA barcoding analysis pipeline
+
+DNA barcoding analysis pipeline designed to perform phylogenetic analysis on raw sanger sequencing data.
+
+## Table of contents
+
+* [Requirements](#Requirements-and-dependencies)
+* [Installation](#Installation)
+* [Example commands](#Example-commands)
+* [Full usage](#Full-usage)
+* [Method](#Method)
+* [Interpeting output](#Interpeting-output)
+* [Acknowledgements](#Acknowledgements)
+* [License](#License)
+
+
+## Requirements and dependencies
+DBA is designed to run on linux-based systems. The pipeline was specifcally tested on Ubuntu 22.04.2 LTS.
+Some tools and most dependencies used during analysis will be installed automatically when the conda environment is being constructed.
+
+The following prerequisites should be installed prior to using DBA
+* Anaconda3
+* Jalview
+	- Note: Jalview should be a system-wide executable as "jalview"
+* Java	
+* MEGA11
+* Python 3.8+
+
+
+## Installation
+
+#### Install dependecies:
+```
+
+```
+
+#### Install DBA:
+```
+git clone https://github.com/mdcjansen/DBA
+cd path/to/DBA
+conda env create -f environment.yml
+chmod a+x DBA.py
+```
+
+#### Allow DBA to be executed from anywhere within the anaconda environment:
+```
+ln -s /path/to/DBA.py /path/to/anaconda3/envs/DBA/bin/DBA
+```
+
+
+## Example commands
+
+#### Activate conda environment for analysis:
+```
+conda activate DBA
+```
+
+#### Display options:
+This command will display all required and optional arguments in the format presented [below](#Full-usage)
+```
+DBA.py
+DBA.py -h "or" --help
+```
+
+#### Minimum required input:
+```
+DBA.py -i <input folder> -n <genbank reference NC_ID> -y <genbank reference YC_ID> -g <genbank outgroup NC_ID>
+```
+
+#### Example input:
+The input below was used to produce the case data available within this repository:
+```
+DBA.py -i case_data -n NC_009065 -y YP_001054869 -g NC_02842
+```
+
+## Full usage
+```
+usage: DBA -i <inputfolder> -n <genbank reference NC_ID> -y <genbank reference YP_ID> -g <genbank outgroup NC_ID> [options]
+
+DBA is designed to automate the process of DNA barcoding by utilising standard Sanger sequencing data and user provided reference gene and protein accession numbers.
+This pipeline obtains nucleic and protein sequences from genbank, followed by chromatogram production in .pdf format by utilising sangerseq viewer.
+Next, BLASTx and BLASTn are run to assess query coverage against the reference gene.
+MUSCLE is used to align the sequences, followed by manual review in jalview and phylogenetic analysis by MEGA.
+
+List of arguments:
+  -h, --help            show this help message and exit
+  -v, --version         Prints program version
+  -i [input folder]     Input folder containing per species folders, a single concatenated fasta file for analysis and a MEGA mao file for phylogenetic analysis
+  -n [genbank reference nucl acc no.]
+                        genbank nucleotide accession number for the gene to be used as reference, usually starts with the identifier NC_
+  -y [genbank reference prot acc no.]
+                        genbank protein accession number for the protein to be used as reference, usually starts with the identifier YC_
+  -g [genbank nucl acc no. for out group]
+                        genbank nucleotide accession number for outgroup gene used during phylogenetic analysis, usually starts with the identifier NC_
+  -o [output folder]    Output directory. 
+                        A default output folder will be produced in the following format will be created if it hasn't been specified:
+                        barcoding_output_current-date_current-time
+  -t [cpu threads]      Maximum amount of threads to be utilised during analysis.
+                        Default: 20
+  -keep []              Keep all files produced during analysis.
+                        Files are stored in 'workdir' folder at the location where this scripthas been executed.
+                        Default: False
+  -rev []               Reverse compliment the fasta input.
+                        Reverse complimented file is saved as an additional file.
+                        Default: False
+```
+
+
+## Method
+DBA requires a specific folder structure in order to perform the analysis.
+A visualisation of this folder structure is given below where case_data is given as `-i` argument:
+
+```
+	.
+	├── ...
+	├── case_data               
+	│	├── sanger.fasta				# FASTA file containing all sanger sequences to be used during analysis
+	│	├── phylo.mao					# MEGA11 generated file used to perform phylogenetic analysis
+	│	├── species_01					# Folder with sequencing data of first species to be analysed
+	│	│	├── sample_01				# Folder containing sanger sequencing data of the first sample
+	│	│	│	├── sanger_data.ab1
+	│	│	│	└── ...
+	│	│	└── ...
+	│	├── species_02					# Folder with sequencing data of second species to be analysed
+	│	│	├── sample_01				# Folder containing sanger sequencing data of the first sample
+	│	│	│	├── sanger_data.ab1
+	│	│	│	└── ...
+	│	│	└── ...
+	│	└── ...
+	└── ...
+```
+
+DBA will perform phylogenetic analysis as presented below:
+1. If specified, DBA will reverse complement the input fasta file
+2. DBA will obtain genbank information on input genbank accession numbers
+3. [sangerseq_viewer](https://github.com/ponnhide/sangerseq_viewer) is utilised to produce a chromatogram for each of the input species found
+4. [BLAST](https://doi.org/10.1016/S0022-2836(05)80360-2) is performed on the input fasta sequence against the input reference sequences
+5. Multple sequence alignment is performed with [MUSCLE](https://doi.org/10.1101/2021.06.20.449169)
+6. DBA initates [Jalview](https://doi.org10.1093/bioinformatics/btp033) for manual review and trimming of alignment data
+7. [MEGA11](https://doi.org/10.1093/molbev/msab120) is used to perform phylogentic analysis and produce a consensus tree
+8. If specified, supplemental data used during analysis is moved to the output folder and the analysis ends.
+
+
+## Interpeting output
+DBA produces a single output folder where all results can be found.
+All procedures during analysis are logged and stored in the barcoding.log file
+
+#### BLAST output
+BLAST results are placed into two tsv files named `blastn.tsv` and `blastx.tsv` for BLASTn and BLASTx results respectively.
+Standard BLAST tabular output format 6 is used to generate the output.
+An example with case data and added header is presented below:
+
+qseqid|sseqid|pident|length|mismatch|gapopen|qstart|qend|sstart|send|evalue|bitscore
+---|---|---|---|---|---|---|---|---|---|---|---|
+RC_EF71414350_EF71414350|NC_009065.1|98.214|672|8|4|13|680|5513|6184|0.0|1171
+RC_EF71414351_EF71414351|NC_009065.1|99.519|624|3|0|5|628|5507|6130|0.0|1136
+RC_EF71414352_EF71414352|NC_009065.1|97.765|671|9|6|5|670|5507|6176|0.0|1151
+RC_EF71414353_EF71414353|NC_009065.1|98.336|661|6|5|10|670|5513|6168|0.0|1155
+RC_EF71414354_EF71414354|NC_009065.1|99.522|628|3|0|6|633|5507|6134|0.0|1144
+
+#### Sangerseq_viewer output
+Sangerseq_viewer produces a chromatogram in pdf format for each species folder present in the input folder.
+These chromatograms display all sequences found inside a particular species folder and present mapped genbank genes.
+An example produced from case data is presented below:
+
+![Screenshot](chromatogram.png)
+
+#### MUSCLE output
+MUSCLE produces multiple alignments files, all presented with the .fa suffix.
+The alignment with the highest column confidence is extracted from the diversified ensemble and opened in jalview to be reviewed by the user.
+Additionally, an html file will be produced for the diversified ensemble containing the letter confidence values of the ensemble.
+
+#### MEGA output
+MEGA generates phylogenetic trees in newick format using user specified parameters specified within the mao file.
+Newick trees and consensus tree are both present in the output folder. Summary and partition text files are also saved within the output.
+
+
+## Acknowledgements
+DBA uses the following tools in the pipeline:
+* [BLAST](https://doi.org/10.1016/S0022-2836(05)80360-2)
+* [Jalview](https://doi.org10.1093/bioinformatics/btp033)
+* [MEGA11](https://doi.org/10.1093/molbev/msab120)
+* [MUSCLE](https://doi.org/10.1101/2021.06.20.449169)
+* [sangerseq_viewer](https://github.com/ponnhide/sangerseq_viewer)
+
+
+## License
+[MIT license](https://opensource.org/license/mit/)
+

licenses/LICENSE__sangerseq_viewer

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+MIT License
+
+Copyright (c) 2022 Yachielab
+
+Permission is hereby granted, free of charge, to any person obtaining a copy
+of this software and associated documentation files (the "Software"), to deal
+in the Software without restriction, including without limitation the rights
+to use, copy, modify, merge, publish, distribute, sublicense, and/or sell
+copies of the Software, and to permit persons to whom the Software is
+furnished to do so, subject to the following conditions:
+
+The above copyright notice and this permission notice shall be included in all
+copies or substantial portions of the Software.
+
+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 AND NONINFRINGEMENT. IN NO EVENT SHALL THE
+AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER
+LIABILITY, WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING FROM,
+OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN THE
+SOFTWARE.