The impact of adaptor selection on genotyping in 2b-RAD studies

Abstract

Population genomic studies have increased in the last decade, showing great potential to understand the evolutionary patterns in a great variety of organisms, mostly relying on RAD sequencing techniques to obtain reduced representations of the genomes. Among them, 2b-RAD can provide further secondary reduction to adjust experimental costs by using base-selective adaptors, although its impact on genotyping is unknown. Here we provide empirical comparisons on genotyping and genetic differentiation when using fully degenerate or base-selective adaptors and assess the impact of missing data. We built libraries with the two types of adaptors for the same individuals and generated independent and combined datasets with different missingness filters (presence in 100%, 75% and 50% of the libraries). Our approach, by exploring locus-by-locus, found 92% of identical genotypes between the two libraries of the same individual when using loci shared among 100% of the libraries, which decreased to 35% when working with loci present in at least 50% of them. We show that missing data is a major source of individual genetic differentiation. The loci discordant by genotyping were in low frequency (7.67%) in all filtered files. Only 0.96% of them were directly attributable to base-selective adaptors reducing heterozygosity. An underestimation of heterozygosity in 6.44% of genotypes was found in libraries generated with fully degenerated adaptors, of which ca. 70% had <10 reads per locus indicating that sufficient read depth should be ensured for a correct genotyping. Base-selective adaptors reduce the number of loci, increase their coverage, and provide a reliable genotyping with no heterozygosity underestimation at a lower sequencing cost. We demonstrate that missing data is a major concern for a correct identification of genetic differentiation, and should not be neglected in genomic studies. Thus, we conclude that 2b-RAD libraries using base-selective adaptors are a robust tool to use in population genomics regardless the target species genome size, promoting their use in future studies.

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“…The quantity and concentration used maximizes the total number of sequences recovered, as shown in previous studies 39 . For this study, we used selective-adaptors (5’-WN-3’) that reduce the number of analysed markers without compromising genetic differentiation 84 . After digestion, ligation and DNA amplification, the quality of the amplified fragment (~ 165 bp) was verified in a 1.8% agarose gel.…”

Genome architecture impacts on reduced representation population genomics

“…Base-selective adaptors can further reduce the number of loci obtained when using 2b-enzymes, and should ideally optimize the costs on population genomic studies without compromising the genomic information (Galià-Camps et al, 2022;Wang et al, 2012). We simulated the effect of using base-selective adaptors with the bash script select_bases_fasta_2.0.sh (Barbanti et al, 2020).…”

“…The number of total and unique loci are key parameters for RADseq studies, as only unique loci will pass the filtering process. Therefore it is of great interest to calculate in advance which is the expected loci number for the study species in order to adapt the number of loci needed for the study and optimize sequencing effort while minimizing missing data (Barbanti et al, 2020;Galià-Camps et al, 2022). Similarly to the percentage of regions in a genomic category, the number of total and unique loci can be best approximated for a new species of interest from the taxon specific regression equation presented here, if an estimation of the genome size is available, since differences have been observed among taxonomic groups.…”

“…Reduced genome sequencing techniques using restriction site digestion enzymes (RAD) are widely used to obtain genome-wide markers of targeted species, rendering population genomic analyses feasible, especially when working with species with big genome sizes or without reference genomes (Guo et al, 2021;Manuzzi et al, 2019;Peterson et al, 2012;Torrado et al, 2020). These methods allow working with many individuals without compromising SNP calling accuracy, since high sequencing depth is required for reliable genotyping (Davey & Blaxter, 2010;Galià-Camps et al, 2022). Restriction enzymes presumably cleave the genome randomly and the resultant fragments are assumed to mirror the genomic structure of the original genome (Davey & Blaxter, 2010;Wang et al, 2012).…”

“…All generated fragments can be sequenced following standard protocols with ligation of fully degenerate adaptors for library building. Nonetheless, this enzyme family allows using base-selective adaptors, which select fragments with desired nucleotides in their sticky ends (Barbanti et al, 2020;Galià-Camps et al, 2022;Wang et al, 2012). This capacity provides to this technique the capacity to further reduce the number of loci, making studies cheaper and therefore allowing to work with species with large genomes, as well as to include many more individuals given a locked budget.…”