Abstract
BackgroundThe irruption of Next-generation sequencing (NGS) and restriction site-associated DNA sequencing (RAD-seq) in the last decade has led to the identification of thousands of molecular markers and their genotyping for refined genomic screening. This approach has been especially useful for non-model organisms with limited genomic resources. Many building-loci pipelines have been developed to obtain robust single nucleotide polymorphism (SNPs) genotyping datasets using a de novo RAD-seq approach, i.e. without reference genomes. Here, the performances of two building-loci pipelines, STACKS 2 and Meyer’s 2b-RAD v2.1 pipeline, were compared using a diverse set of aquatic species representing different genomic and/or population structure scenarios. Two bivalve species (Manila clam and common edible cockle) and three fish species (brown trout, silver catfish and small-spotted catshark) were studied. Four SNP panels were evaluated in each species to test both different building-loci pipelines and criteria for SNP selection. Furthermore, for Manila clam and brown trout, a reference genome approach was used as control.ResultsDespite different outcomes were observed between pipelines and species with the diverse SNP calling and filtering steps tested, no remarkable differences were found on genetic diversity and differentiation within species with the SNP panels obtained with a de novo approach. The main differences were found in brown trout between the de novo and reference genome approaches. Genotyped vs missing data mismatches were the main genotyping difference detected between the two building-loci pipelines or between the de novo and reference genome comparisons.ConclusionsTested building-loci pipelines for selection of SNP panels seem to have low influence on population genetics inference across the diverse case-study scenarios here studied. However, preliminary trials with different bioinformatic pipelines are suggested to evaluate their influence on population parameters according with the specific goals of each study.
Highlights
The irruption of Next-generation sequencing (NGS) and restriction site-associated DNA sequencing (RAD-seq) in the last decade has led to the identification of thousands of molecular markers and their genotyping for refined genomic screening
Preliminary trials with different bioinformatic pipelines are suggested to evaluate their influence on population parameters according with the specific goals of each study
RAD-seq methods require specific library preparation protocols, which exploit the ability of Restriction Enzymes (REs) to cut at specific genomic targets rendering a collection of fragments representative of a genome fraction to be compared among samples
Summary
The irruption of Next-generation sequencing (NGS) and restriction site-associated DNA sequencing (RAD-seq) in the last decade has led to the identification of thousands of molecular markers and their genotyping for refined genomic screening. By harnessing the possibilities of NGS, diverse reduced-representation genome sequencing approaches, useful to identify and genotype thousands of markers for genomic screening, were suggested and quickly became popular [4, 5] One of these approaches is the restriction site-associated DNA sequencing (RADseq), currently in a more mature phase, which includes different methods (e.g. ddRAD-seq, ezRAD-seq, 2bRAD-seq) whose performances have been compared using simulations and real data [6]. RAD-seq methods require specific library preparation protocols, which exploit the ability of Restriction Enzymes (REs) to cut at specific genomic targets rendering a collection of fragments representative of a genome fraction to be compared among samples These collections can be screened to identify and genotype a variable number of single nucleotide polymorphisms (SNPs) depending on the goals of the study for population genomics, linkage mapping or genome wide association studies, among others. This method has the advantages of simple library preparation, short-reads to be sequenced (single-end 50 bp) and, as other methods, the number of loci can be adjusted both using REs with different recognition site frequency or by fixing nucleotides in the adaptors during library construction (i.e. selective-base ligation) [7, 8]
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