MycoGene: A workflow for extracting specific gene sequences from WGS Illumina paired-end short reads

Description

MycoGene is an innovative workflow for Multi-Locus Sequence Typing (MLST) analysis of fungal organisms, designed to streamline your research. By replacing traditional PCR and Sanger sequencing with WGS and simple bioinformatics, MycoGene significantly reduces time and effort in the lab.

This workflow performs quality controls, generates de novo assemblies, and extracts target gene sequences. The FASTA output files are compatible with various downstream analytical tools for build phylogenetic trees and visualize alignments.

Among its most useful applications are taxonomic profiling, species identification, and finding mutations linked to antifungal resistance. MycoGene shines particularly in non-model fungi where complete genome annotation is lacking, making it a straight solution for identifying mutation in specific genes when annotate whole-genome variants is unpractical.

Azole resistance mutations in A. fumigatus CPY51 gene

For using MycoGene to identify mutation in Aspergillus fumigatus CPY51 gene use branch Afum_CPY51 git clone --single-branch --branch Afum_CPY51 https://github.com/CDCgov/MycoGene.git

Inputs:

1. samples-paths.txt, Plain text file that contains the samples name and paths for raw reads, separated by :. One sample per row, no header.
   Example: Sample1:/my-path/sample1_R1.fastq.gz:/my-path/sample1_R2.fastq.gz Sample2:/my-path/sample2_R1.fastq.gz:/my-path/sample2_R2.fastq.gz

2. gene_list, Plain tex file that contains the list of target genes, it's recommended to add prefix organis/project related. Example: Crypto_CAP59 Crypto_GPD1 Crypto_LAC1

3. queries, Directory that contains Fasta files for each target gene. File name must match the gene name in gene_list file

Outputs:

A directory for each gene that contains the following files:

1. aln_gene_output.fasta, Fasta file that contains aligned nucleotide sequence of the gene of interest for all samples.
2. aln_protein_output.fasta, Fasta file that contains aligned aminoacid sequence of the protein of interest for all samples.
3. aln_wnoncoding_gene_output.fasta, Fasta file that contains aligned nucleotide sequence of the gene of interest plus a 500 nucleotides upstream of the coding gene for all samples.Upstream region include the gene promotor for identifing the tandem repeats (TR34, TR46)
4. QC_report.txt, Tabular text file with blastn hits information.
   
   Note: Review QC report for the following quality metrics:

| QC Metric | Threshold | | ------------------------------- |-------------------------------------| | Percentage of identity (pident) | > 95 % | | Length | Similar to the query sequence length|

Requirements

• FaQCs (https://github.com/LANL-Bioinformatics/FaQCs)
• SPAdes (https://github.com/ablab/spades)
  
• blast (https://www.ncbi.nlm.nih.gov/books/NBK569861/)
  
• samtools (https://bioconda.github.io/recipes/samtools/README.html)
  
• Clustal omega (https://github.com/GSLBiotech/clustal-omega)
  
• mView (https://sourceforge.net/projects/bio-mview/files/bio-mview)

Running MycoGene

1. Ensure your inputs are placed in the same directory as the MycoGene_blastn_v1.1.sh and Mycogene_multiquery_v2.sh scripts.
2. nohup bash Mycogene_multiquery_v2.sh

Workflow

Roadmap

Some potential enhancements and use cases: 1. Use multiples genes of interest as query. Potential use for specie identification with more than one marker. Ex. Genes TEF1, rDNA, RPB2 for Fusarium species identification. 2. To add BLAST type as a user selection parameter (tblastn or blastn), to perform the analysis using a nucleotide sequence as query. Potential use for recovering noncoding sequence region. Ex. Promotor sequence and tandem repeats for CYP51 gene for Aspergillus. .

Citations

If you use MycoGene in your work, please consider this citing repository https://github.com/CDCgov/MycoGene

CDCgov GitHub Organization Open Source Project

General disclaimer This repository was created for use by CDC programs to collaborate on public health related projects in support of the CDC mission. GitHub is not hosted by the CDC, but is a third party website used by CDC and its partners to share information and collaborate on software. CDC use of GitHub does not imply an endorsement of any one particular service, product, or enterprise.

Access Request, Repo Creation Request

• CDC GitHub Open Project Request Form [Requires a CDC Office365 login, if you do not have a CDC Office365 please ask a friend who does to submit the request on your behalf. If you're looking for access to the CDCEnt private organization, please use the GitHub Enterprise Cloud Access Request form.]

Related documents

• Open Practices
• Rules of Behavior
• Thanks and Acknowledgements
• Disclaimer
• Contribution Notice
• Code of Conduct

Overview

Describe the purpose of your project. Add additional sections as necessary to help collaborators and potential collaborators understand and use your project.

Public Domain Standard Notice

This repository constitutes a work of the United States Government and is not subject to domestic copyright protection under 17 USC § 105. This repository is in the public domain within the United States, and copyright and related rights in the work worldwide are waived through the CC0 1.0 Universal public domain dedication. All contributions to this repository will be released under the CC0 dedication. By submitting a pull request you are agreeing to comply with this waiver of copyright interest.

License Standard Notice

The repository utilizes code licensed under the terms of the Apache Software License and therefore is licensed under ASL v2 or later.

This source code in this repository is free: you can redistribute it and/or modify it under the terms of the Apache Software License version 2, or (at your option) any later version.

This source code in this repository is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the Apache Software License for more details.

You should have received a copy of the Apache Software License along with this program. If not, see http://www.apache.org/licenses/LICENSE-2.0.html

The source code forked from other open source projects will inherit its license.

Privacy Standard Notice

This repository contains only non-sensitive, publicly available data and information. All material and community participation is covered by the Disclaimer and Code of Conduct. For more information about CDC's privacy policy, please visit http://www.cdc.gov/other/privacy.html.

Contributing Standard Notice

Anyone is encouraged to contribute to the repository by forking and submitting a pull request. (If you are new to GitHub, you might start with a basic tutorial.) By contributing to this project, you grant a world-wide, royalty-free, perpetual, irrevocable, non-exclusive, transferable license to all users under the terms of the Apache Software License v2 or later.

All comments, messages, pull requests, and other submissions received through CDC including this GitHub page may be subject to applicable federal law, including but not limited to the Federal Records Act, and may be archived. Learn more at http://www.cdc.gov/other/privacy.html.

Records Management Standard Notice

This repository is not a source of government records, but is a copy to increase collaboration and collaborative potential. All government records will be published through the CDC web site.

Additional Standard Notices

Please refer to CDC's Template Repository for more information about contributing to this repository, public domain notices and disclaimers, and code of conduct.

SHARE IT Act Metadata

• Organization: NCEZID/DFWED/MDB
• Contact Email: ncezid_shareit@cdc.gov