Helping decision making for reliable and cost‐effective 2b‐RAD sequencing and genotyping analyses in non‐model species

Abstract

High-throughput sequencing has revolutionized population and conservation genetics. RAD sequencing methods, such as 2b-RAD, can be used on species lacking a reference genome. However, transferring protocols across taxa can potentially lead to poor results. We tested two different IIB enzymes (AlfI and CspCI) on two species with different genome sizes (the loggerhead turtle Caretta caretta and the sharpsnout seabream Diplodus puntazzo) to build a set of guidelines to improve 2b-RAD protocols on non-model organisms while optimising costs. Good results were obtained even with degraded samples, showing the value of 2b-RAD in studies with poor DNA quality. However, library quality was found to be a critical parameter on the number of reads and loci obtained for genotyping. Resampling analyses with different number of reads per individual showed a trade-off between number of loci and number of reads per sample. The resulting accumulation curves can be used as a tool to calculate the number of sequences per individual needed to reach a mean depth ≥20 reads to acquire good genotyping results. Finally, we demonstrated that selective-base ligation does not affect genomic differentiation between individuals, indicating that this technique can be used in species with large genome sizes to adjust the number of loci to the study scope, to reduce sequencing costs and to maintain suitable sequencing depth for a reliable genotyping without compromising the results. Here, we provide a set of guidelines to improve 2b-RAD protocols on non-model organisms with different genome sizes, helping decision-making for a reliable and cost-effective genotyping.

Achieving high-quality ddRAD-like reference catalogs for non-model species: the power of overlapping paired-end reads

“…There is an ever-growing number of studies trying to overcome issues produced by RAD experiments and data (e.g. Barbanti et al, 2020;Chow et al, 2019;LaCava et al, 2020;Paris et al, 2017;Shafer et al, 2017). However, the issues caused by the technique are highly variable and seem to be specific to each experiment (DaCosta & Sorenson, 2014).…”

“…Low coverage results in decreased ability to confidently detect heterozygotes, affecting the analysis involving individual genotypes (Barbanti et al, 2020;Chow et al, 2019). This should specially be avoided when building the set of R2SCOs for a species, as the evaluation of heterozygosity as well as the potential technical issues regarding some loci will be crucial to help planning larger scale analyses (that could use shorter reads) and therefore would decrease issues related to genotyping at the population level.…”

Genomic outcomes from diverse SNP panels obtained with different de novo building-loci pipelines in a varied species context: Can pipelines be influencing biological interpretations?

“…This genomic information should be considered to make the best technical decisions. Without reference genome, different REs can be tested if the budget allows it (see Box 1 in [8]), to improve the percentage of reads to build up con dent loci. In the same way, the different performances of software and bioinfomatic pipelines might depend on the species and its genomics context.…”

“…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 xing nucleotides in the adaptors during library construction (i.e. selective-base ligation) [7,8].…”

“…Genomic laboratory protocols have been set up and optimized through years by introducing modi cations on RAD-seq to achieve the better results using different lab protocols (see Fig. 5 in [8]).…”