RADSex: a computational workflow to study sex determination using Restriction Site-Associated DNA Sequencing data

Abstract

The study of sex determination and sex chromosome organisation in non-model species has long been technically challenging, but new sequencing methodologies are now enabling precise and high-throughput identification of sex-specific genomic sequences. In particular, Restriction Site-Associated DNA Sequencing (RAD-Seq) is being extensively applied to explore sex determination systems in many plant and animal species. However, software designed to specifically search for sex-biased markers using RAD-Seq data is lacking. Here, we present RADSex, a computational analysis workflow designed to study the genetic basis of sex determination using RAD-Seq data. RADSex is simple to use, requires few computational resources, makes no prior assumptions about type of sex-determination system or structure of the sex locus, and offers convenient visualization through a dedicated R package. To demonstrate the functionality of RADSex, we re-analyzed a published dataset of Japanese medaka, Oryzias latipes , where we uncovered a previously unknown Y chromosome polymorphism. We then used RADSex to analyze new RAD-Seq datasets from 15 fish species spanning multiple systematic orders. We identified the sex determination system and sex-specific markers in six of these species, five of which had no known sex-markers prior to this study. We show that RADSex greatly facilitates the study of sex determination systems in non-model species and outperforms the commonly used RAD-Seq analysis software STACKS.RADSex in speed, resource usage, ease of application, and visualization options. Furthermore, our analysis of new datasets from 15 species provides new insights on sex determination in fish.

The rise and fall of the ancient northern pike master sex-determining gene

“…Raw RAD-seq reads were demultiplexed with the process_radtags.pl script from stacks version 1.44 ( Catchen et al, 2013 ) with all parameters set to default. Demultiplexed reads for each species were analyzed with the RADSex computational workflow ( Feron et al, 2020a ) using the radsex software version 1.1.2 (10.5281/zenodo.3775206). After generating a table of markers depth with process , the distribution of markers between sexes was computed with distrib and markers significantly associated with phenotypic sex were extracted with signif using a minimum depth of 10 (−d 10) for both commands and with all other settings at the default.…”

The genome of the bowfin (Amia calva) illuminates the developmental evolution of ray-finned fishes

“…Thus, sex determination in bow n is either polyfactorial, too small to be detected using Pool-Seq, and/or based on environmental effects. Notably, no genomic sex differentiation loci have been found using similar approaches for both spotted gar 38 and the more distantly related sterlet sturgeon 39 . Genetic sex determination mechanisms thus remain elusive for nonteleost ray-nned shes, if they exist.…”

“…RNA was extracted from each individual tissue (Qiagen RNEasy kit), quanti ed (ThermoFisher NanoDrop and Agilent BioAnalzyer), and pooled in equal amounts prior to library barcoding. Sequencing reads from NovaSeq 6000 were quality-trimmed using Trimmomatic v0 38.…”

Limited evidence of a genetic basis for sex determination in the common creek chub, Semotilus atromaculatus

“…Initial analysis of sex-associated markers in the creek chub genome was performed using RADSex, a computational workflow intended as an "all-in-one" GSD detection program and visualizer, with the assistance of the RStudio package sgtr (Feron et al 2021). RADSex treats all markers as unassociated genotypes, rather than alleles of the marker, and searches for markers that are found in one sex exclusively, at a statistically significant level (Feron et al 2021). Thus, it is theoretically able to detect heteromorphic or homomorphic sex chromosomes, or polygenic sex determination loci, using only one workflow.…”