Cassava Bioinformatics Platform: QTL analysis Pipeline

Pipeline for QTL analysis on bi-parental families in Cassava

Authors: Vianey Barrera-Enriquez and Camilo E. Sanchez-Sarria

The pipeline has four main steps:

1. Single QTL mapping
2. Multiple QTL mapping (MQM) (in progress)
3. Customized plots (LOD profiles, Missing genotypes, etc)
4. Annotation of results

1. Single QTL Mapping

This code performs QTL (Quantitative Trait Locus) analysis using Rqtl package. It computes single QTLs, LOD/Bayes intervals, scores, and effects using a mapqtl input format.

Usage

single_qtl(dir, dircross, dirfun, locfile, mapfile, phenofile, prefixResults, ncores, step = 0.5, off.end = 0, error.prob = 0.001, alpha = 0.1, n.perm = 1000, map.function = "kosambi", stepwidth = "fixed", model_scanone = "normal")

Arguments

1.1. For Single QTL function:

• dir: Directory to save results.
• dircross: Directory where data is located. It can be empty if the cross files include the path and name.
• dirfun: Directory were functions for plotting are located.
• locfile: .loc file name. It can include the path.
• mapfile: .map file name. It can include the path.
• cphenofile: .qua file name. It can include the path.
• prefixResults: The prefix that the results will have.
• ncores: Number of cores for permutation test.

1.2. For Single QTL function (scanone function part):

• step: Step size in cM used for interval mapping (default = 0.5).
• off.end: A value added to each end of the chromosome, to extend the genetic map (default = 0).
• error.prob: The probability of a genotyping error (default = 0.001).
• alpha: The significance level to use for peak detection (default = 0.1).
• n.perm: Number of permutations to use for significance testing (default = 1000).
• map.function: The genetic map function to use (default = "kosambi").
• stepwidth: Method used to compute step size. Can be "fixed" or "cov" (default = "fixed").
• model_scanone: Model used in the single-QTL scan (default = "normal". Options = "normal", "binary" or "poisson").

Details

The data is read using the read.cross() function. The cross data is in mapqtl format, which includes .map, .loc, and .qua files with the genetic map, markers, and phenotypes information, respectively. The calc.genoprob() function is used to calculate the probabilities of each individual's genotype given their observed phenotype data. The scanone() function is used to perform interval mapping using the Expectation-Maximization (EM) algorithm, which estimates the genetic effects of the markers in the cross. A permutation test is conducted using scanone() to compute the genome-wide significance threshold. Peaks are identified as locations in the genome where the genetic effect of the markers is above the genome-wide significance threshold.

Output

In progess

Dependencies

• qtl
• tidyverse
• snow

2. Multiple QTL mapping (MQM)

In progress

3. Customized plots

In progress

4. Annotation of results

This code annotates the gen that overlaps and/or contain the significant QTLs obtained from the rqtl results.

Usage

QTL_Annotation(Wdir, Ddir, name, wdyw, annot, gff, version, recursive)

Arguments

• Wdir: Name of the directory that contains the GAPIT results. For example: home/user/folder.
• Ddir: Directory where is located the annotation files (annot, GFF files).
• name: Enter the path or the name of file names to look for. For example: QTL_LOD_Intervals.
• wdyw: Enter what are you looking for to annotate (Options: CDS, five_prime_UTR, gene, mRNA, three_prime_UTR).
• annot: Annotation details of the genes. txt file from the genome version used for alignment.
• gff: gff3 file from the genome version used for alignment.
• version: You can choose between the genome of reference version 6.1 or 8.1 (Options: 6.1 or 8.1).
• recursive: A Boolean string that determines if the function should perform a recursive search or not (Default = F).

Details

In progress

Examples

# Set arguments
Ddir <- "D:/OneDrive - CGIAR/Cassava_Bioinformatics_Team/00_Data/"
wdyw <- "gene"
annot <- "Mesculenta_305_v6.1/Mesculenta_305_v6.1.annotation_info.txt"
gff <- "Mesculenta_305_v6.1/Mesculenta_305_v6.1.gene.gff3"
version <- "6.1"

# If recursive
Wdir <- "D:/OneDrive - CGIAR/Cassava_Bioinformatics_Team/01_ACWP_F1_Metabolomics/02_QTL_Analysis/CM8996/Everything/"
name <- "LodIntervals"
recursive <- "T"

# Non-recursive
Wdir <- "D:/OneDrive - CGIAR/Cassava_Bioinformatics_Team/01_ACWP_F1_Metabolomics/02_QTL_Analysis/CM8996/Significant_ones/"
name <- "QTL_results_heritability.csv"
recursive <- "F"


# Run function
QTL_Annotation(Wdir, Ddir, name, wdyw, annot, gff, version, recursive)

Output

A single CSV file containing relevant gene information plus QTLs' LOD, P-values, traits, and effects.

Dependencies

• tidyverse
• openxlsx

5. Boxplot of significant markers: genotypes vs. phenotype

This R function generates a boxplot for a given SNP. The function takes as inputs a CSV file with the phenotype values and a list of SNPs. The function can also receive an optional CSV file to add extra information about the samples to the plot (categories, family, ect.) The output of the function is a PDF file with the plot.

Usage

QTL_Boxplot(outputname, dir, phenofile, genofile, snp_list_file, labelfile = NULL)

Arguments

• outputname: (required) character string with the base name for the output file.
• dir: (required) character string with the directory where the output file will be saved.
• phenofile: (required) character string with the name of the phenotype file in tabular format. The first column should contain the sample names, and the rest of the columns should contain the phenotypes.
• genofile: (required) character string with the name of the genotype file in hapmap format.
• snp_list_file: (required) character string with the name of the CSV file with three columns:
  • Column 01 - Name: SNPS. List of SNPS to plot. The name should be the same as in the genofile data.
  • Column 02 - Name: trait. Name of the trait as in the phenofile data.
  • Column 03 - Name: xlabel. Name of the trait to be included as a label.
• labelfile: (optional) character string with the name of the CSV file with two columns:
  • Column 01 - Name: Taxa. Sample names.
  • Column 02 - Name: label. Label or category to add to the plot.

Details

The function loads all the necessary packages and data files. It then checks for the presence of an optional file with sample labels and prepares the data for the boxplot. The function generates a boxplot for each SNP specified in the input CSV file. It uses ggplot2 package to generate the plot, with the genotypes on the x-axis and the phenotype on the y-axis. The function adds a label to the x-axis to indicate the trait being plotted. If labelfile is provided, it adds extra information about the samples, coloring the data by the levels in the provided file.

Example

# Generate boxplot without extra labels
QTL_Boxplot("outputname", ".path/to/save/plots/", "phenotype.csv", "genotype.hmp", "snp_list.csv")

# Generate boxplot with extra labels
QTL_Boxplot("outputname", ".path/to/save/plots/", "phenotype.csv", "genotype.hmp", "snp_list.csv", "labelfile.csv")

Output

A single PDF file containing the boxplot of the SNPs.

Dependencies

• tidyverse
• tibble
• dplyr
• janitor
• ggplot2
• Biostrings
• hrbrthemes
• forcats
• ggsignif
• RColorBrewer

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Contact

For questions or feedback about this pipeline, please contact Vianey Barrera-Enriquez at [email protected].