Web-based Shiny App for automatic codon optimization and analysis based on codon usage rules in non-Caenorhabditis nematode species, including: Strongyloides species, Pristionchus pacificus, Nippostrongylus brasiliensis, Brugia malayi, as well as any other species of interest via custom codon usage rules provided by users.
- General Information
- App Setup & Deployment
- App Features
- App Methods
- Examples of App Use
- Sources
- License
- Authors
This repository contains the infrastructure for generating a Shiny web application. The app is deployed via Shinyapps.io but can also be run locally. See App Setup and App Features sections below for additional details.
For more information please see the G3 paper associated with this project
To access a stable deployment of the Wild Worm Codon Adapter Web App, please visit: https://hallemlab.shinyapps.io/Wild_Worm_Codon_Adapter/
To run the latest version locally from GitHub, use the following command in R/RStudio:
library(shiny)
shiny::runGitHub(repo = 'Wild_Worm_Codon_Adapter', username = 'HallemLab')
To run a specific release locally use the following commands in R/RStudio:
-
For PCs --
library(shiny)
shiny::runUrl('https://github.com/HallemLab/Wild_Worm_Codon_Adapter/archive/<RELEASE_VERSION>.zip') -
For Macs --
library(shiny)
shiny::runUrl('https://github.com/HallemLab/Wild_Worm_Codon_Adapter/archive/<RELEASE_VERSION>.tar.gz')
Please note: the download step for runURL/runGitHub may take a substantial amount of time. We recommend downloading this archive and running the application locally.
The Wild Worm Codon Adapter Web Tool adapts and automates the process of codon adaptation for a selection of non-Caenorhabditis nematode species, including: Strongyloides species, Nippostrongylus brasiliensis, Brugia malayi, Pristionchus pacificus, as well as Caenorhabditis elegans. It also permits codon optimization via user-provided custom optimal codon sets. Furthermore, this tool enables users to perform bulk calculations of codon adaptiveness relative to species-specific codon usage rules.
The app has two usage modes:
- Optimization Mode: This tab optimizes genetic sequences for expression in Strongyloides species, N. brasiliensis, B. malayi, P. pacificus, and C. elegans, as well as user-provided optimal codon sets.
It accepts either nucleotide or single-letter amino acid sequences, and will generate an optimized nucleotide sequence with and without the desired number of introns. Users may choose between using canonical Fire lab synthetic introns, PATC-rich introns, P. pacificus native introns, or a custom set of user-provided introns. Users may input sequences for optimization using the text box provided, or may upload sequences as .fasta/.gb/.txt files.
Optimized sequences with or without artificial introns may be downloaded as plain text (.txt) files.
- Analysis Mode: For user-provided genes/sequences, this tab reports the fractional GC content, coding sequence, and codon optimization relative to the codon usage weights of:
- highly expressed S. ratti transcripts (Mitreva et al 2006)
- highly expressed C. elegans genes (Sharp and Bradnam, 1997)
- highly expressed N. brasiliensis genes (Eccles et al 2018)
- highly expressed B. malayi genes (Han et al, 2020)
- highly expressed P. pacificus genes (Han et al, 2020)
To analyze transgenes, coding sequences can be provided via a text box. To analyze native genes, stable gene or transcript IDs with prefixes "SSTP", "SRAE", "SPAL", "SVE", "NBR", "Bma", "Ppa", or "WB" can be provided either through direct input via the appropriate textbox, or in bulk as a comma separated (CSV) text file. Users may also provide a C. elegans gene name, provided it is prefaced with the string "Ce-", or C. elegans stable transcript IDs as is. Finally, users may direcly provide coding sequences for analysis, either as a 2-column CSV file listing sequence names and coding sequences, or a FASTA file containing named coding sequences.
Users may download an excel file containing fractional GC content values, codon adaptation indeces, and coding sequences for the user-provided genes.
The primary non-responsive data inputs to the Wild Worm Codon Adapter App are CSV files containing the following information:
- Codon frequency rates and relative adaptiveness values for S. ratti, N. brasiliensis, B. malayi, P. pacificus, and C. elegans
- Optimal codon lookup table for Strongyloides spp, N. brasiliensis, B. malayi, P. pacificus, and C. elegans
- Custom optimal codon lookup table (2 columns: single-letter amino acid symbols and corresponding 3-letter optimal codon sequences; one optimal codon per amino acid)
- Custom intron list (fasta file containing a maximum of 3 introns; intron sequences should begin/end with canonical 5'-GTAG-3' splice recognition sequences)
These data are loaded by the Shiny server function and used to calculate CAI values and optimize sequences.
The codon usage patterns of highly expressed genes are thought to correlate with higher protein expression ( Sharp and Li, 1987 , Plotkin and Kudla, 2011 ). Thus, codon frequency rates from highly expressed genes are used.
Codon frequency rates for Strongyloides species are based on highly expressed S. ratti transcripts (50 most abundant expressed sequence tag clusters). Specifically, codon usage rules were generated by calculating the frequency for each codon from count data published in Mitreva et al (2006); frequency values were manually checked against published frequency values.
Codon frequency rates for C. elegans were based on highly expressed C. elegans gene count data published in Sharp and Bradnam (1997).
Codon frequency rates for N. brasiliensis were calculated from coding sequences of highly expressed N. brasiliensis genes (10% highest RNA-seq expression values across all samples); RNA-seq data was downloaded from WormBase ParaSite, based on data originally published in Eccles et al (2018), and Chandler et al (2017). Codon frequency rates for highly expressed B. malayi and P. pacificus genes (average frequency bins 8-11, ~10% highest expressing genes) are from Han et al (2020); raw codon frequency data were graciously provided by Dr. Wen-Sui Lo and Dr. Ralf Sommer.
The relative adaptiveness values for every possible codon were generated as follows. Individual codons were scored by calculating their relative adaptivness: (the frequency that codon "i" encodes amino acid "AA") / (the frequency of the codon most often used for encoding amino acid "AA"). Optimal codons for these species were defined as the codon with the highest relative adaptiveness value for each amino acid.
User-provided custom optimization rules: In addition to the optimization rules provided by the application, users may also provide a custom set of optimal codons. In this case, users may upload a CSV file containing 2 columns listing single-letter amino acid symbols and the corresponding 3-letter optimal codon sequence, using the provided UI interface. Only one optimal codon should be provided per amino acid; stop codons should be designated using the '*' symbol. This custom optimal codon lookup table will be applied during codon optimization; CAI values will not be calculated.
In all cases, codon optimzation is performed by replacing non-optimal codons with optimal codons.
Sequences (both original and optimized) are scored by calculating the Codon Adaptation Index: the geometric average of relative adaptiveness of all codons in the gene sequence ( Sharp and Li 1987, Jansen et al 2003). The CAI is calculated via the seqinr library using a multi-species relative adaptiveness table (see above).
The fraction of G+C bases of the nucleic acid sequences. Calculated using the seqinr library.
Incorporating introns into cDNA sequences can signficiantly increase gene expression in nematode species (Crane et al 2019, Han et al 2020, Junio et al 2008, Li et al 2011).
In Optimize Sequences mode, users may input a desired number of introns, up to a maximum of three unique introns. The Fire lab established three unique introns, spaced equidistantly within a gene, as canon (Fire Lab Vector Kit 1995); this configuration is thus set as default, and is recommended.
Intron sequences and insertion order are either the three canonical Fire lab synthetic introns established by the Fire lab, P. pacificus native introns, PATC-rich introns (smu-2 introns 3-5) that enhance germine expression of transgenes in C. elegans, or custom user-provided intron sequences (Fire Lab Vector Kit 1995, Han et al 2020, Aljohani et al 2020). All built-in introns sequences are bracketed with canonical GT...AG splice recognition sequences (Shapiro and Senapathy 1987, Blumenthal and Steward 1997, Wheeler et al 2020). Users may either select the desired built-in intron sequence source using the dropdown menu provided, or upload a fasta file containing up to three custom intron sequences.
This app first divides the optimized cDNA sequence at 3 predicted intron insertion sites spaced approximately equidistantly. Users may choose to further refine the insertion site locations by identifying the closest conserved invertebrate exon splice sites (‘AG^G’, ‘AG^A’)( Shapiro and Senapathy, Blumenthal and Steward). For all insertion sites, '^' symbol indicates the exact insertion site.
Once hypothetical intron insertion sites have been identified, the application inserts the user-specified number of introns, using the 5’ insertion site first and continuing in the 3’ direction. In C. elegans, the location of the intron site influences the degree of intron-mediated enhancement, such that a single 5′-intron is more effective than a single 3′-intron (Crane et al 2019, Aljohani et al 2020). Therefore when only 1 or 2 introns are desired, 3 possible intron insertion sites are identified and filled as needed, starting from the 5′ site.
- Shiny - UI framework
- Wormbase ParaSite - GeneIDs and coding sequences
- Seqinr - Utilities for calculating Codon Adaptation Index
- Codon Usage Patterns:
- Strongyloides spp: Mitreva et al (2006)
- Pristionchus pacificus: Han et al (2020)
- Brugia malayi: Han et al (2020)
- Nippostrongylus brasiliensis: WormBase ParaSite, Eccles et al (2018)
- C. elegans: Sharp and Bradnam, 1997
- Intron Sequences:
- Canonical Fire lab artificial introns: Fire Lab Vector Kit 1995
- PATC-rich introns: Aljohani et al (2020)
- P. pacificus native introns: Han et al (2020)
- Intron/Exon Splice Sites: Shaprio and Senapathy (1987) and Blumenthal and Steward (1997)
This project is licensed under the MIT License.