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

Abstract

Reduced representation libraries present an opportunity to perform large scale studies on non-model species without the need for a reference genome. Methods that use restriction enzymes and fragment size selection to help obtain the desired number of loci -such as ddRAD -are highly flexible and therefore suitable to different types of studies. However, a number of technical issues are not approachable without a reference genome, such as size selection reproducibility across samples and coverage across fragment lengths. Moreover, identity thresholds are usually chosen arbitrarily in order to maximize the number of SNPs considering arbitrary parameters. We have developed a strategy to identify de novo a set of reduced-representation single-copy orthologs (R2SCOs). Our approach is based on overlapping reads that recreate original fragments and add information about coverage per fragment size. A further in silico digestion step limits the data to well covered fragment sizes, increasing the chance of covering the majority of loci across different individuals. By using full sequences as putative alleles, we estimate optimal identity thresholds from pairwise comparisons. We have demonstrated our full workflow with data from five sea turtle species. Locus numbers were similar across all species, even at increasing phylogenetics distances. Our results indicated that sea turtles have in general very low levels of heterozygosity. Our approach produced a high-quality set of reference loci, eliminating a series of biological and experimental biases that can strongly affect downstream analysis, and allowed us to explore the genetic variability within and across sea turtle species.

New genetic insights about hybridization and population structure of hawksbill and loggerhead turtles from Brazil

“…The initial selection of nDNA markers was performed using a reduced genomic dataset produced via ddRAD. As a preliminary analysis to help establishing a standardized ddRAD protocol for sea turtles (Driller et al 2020), one individual of each parental species ( E. imbricata and C. caretta) and one F1 Ei × Cc hybrid individual were used for ddRAD library construction. The sequencing library was generated by digesting the genomic DNA using the restriction enzymes Nde I and Mlu CI with subsequent ligation of Illumina adapters followed by a 10-cycle Polymerase Chain Reaction (PCR) for completeness of sequencing adapters, as described in Peterson et al (2012).…”

New Genetic Insights About Hybridization and Population Structure of Hawksbill and Loggerhead Turtles From Brazil

“…The initial selection of nDNA markers was performed using a reduced genomic dataset produced via ddRAD. As a preliminary analysis to help establishing a standardized ddRAD protocol for sea turtles (Driller et al 2020), one individual of each parental species (E. imbricata . CC-BY-NC-ND 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.…”

Inbreeding depression depends on the body size and environmental conditions in a threatened songbird, the aquatic warblerAcrocephalus paludicola

“…2016). First, because the genome of the aquatic warbler is not yet available, we constructed a reference genomic library according to the Reduced-Representation Single-Copy Orthologs (R2SCOs) method, following Driller et al . (2021), with modifications (see below).…”