Experimental validation of in silico predicted RAD locus frequencies using genomic resources and short read data from a model marine mammal

Abstract

BackgroundRestriction site-associated DNA sequencing (RADseq) has revolutionized the study of wild organisms by allowing cost-effective genotyping of thousands of loci. However, for species lacking reference genomes, it can be challenging to select the restriction enzyme that offers the best balance between the number of obtained RAD loci and depth of coverage, which is crucial for a successful outcome. To address this issue, PredRAD was recently developed, which uses probabilistic models to predict restriction site frequencies from a transcriptome assembly or other sequence resource based on either GC content or mono-, di- or trinucleotide composition. This program generates predictions that are broadly consistent with estimates of the true number of restriction sites obtained through in silico digestion of available reference genome assemblies. However, in practice the actual number of loci obtained could potentially differ as incomplete enzymatic digestion or patchy sequence coverage across the genome might lead to some loci not being represented in a RAD dataset, while erroneous assembly could potentially inflate the number of loci. To investigate this, we used genome and transcriptome assemblies together with RADseq data from the Antarctic fur seal (Arctocephalus gazella) to compare PredRAD predictions with empirical estimates of the number of loci obtained via in silico digestion and from de novo assemblies.ResultsPredRAD yielded consistently higher predicted numbers of restriction sites for the transcriptome assembly relative to the genome assembly. The trinucleotide and dinucleotide models also predicted higher frequencies than the mononucleotide or GC content models. Overall, the dinucleotide and trinucleotide models applied to the transcriptome and the genome assemblies respectively generated predictions that were closest to the number of restriction sites estimated by in silico digestion. Furthermore, the number of de novo assembled RAD loci mapping to restriction sites was similar to the expectation based on in silico digestion.ConclusionsOur study reveals generally high concordance between PredRAD predictions and empirical estimates of the number of RAD loci. This further supports the utility of PredRAD, while also suggesting that it may be feasible to sequence and assemble the majority of RAD loci present in an organism’s genome.Electronic supplementary materialThe online version of this article (10.1186/s12864-019-5440-8) contains supplementary material, which is available to authorized users.

Genetic variability of the 16S rRNA gene of Nocardia brasiliensis, the most common causative agent of actinomycetoma in Latin America and the Caribbean

“…To broaden and deepen the genetic variability found, highly discriminatory studies such as enzyme restriction could be performed on other constitutive genes in N. brasiliensis. In silico enzyme restriction studies are useful for expanding the number of enzymes available in the assay and thus making an appropriate choice of enzymes [24][25][26] , but they are also useful for reducing costs and materials in the experiment because they have a high coincidence with the results shown in vitro [26][27][28][29] .…”

Reduced metagenome sequencing for strain-resolution taxonomic profiles

“…As an alternative to do a full shotgun metagenomic sequencing, the use of restriction enzymes to reduce the genomic sequence space has also been employed to investigate microbial communities [ 7 – 9 ]. The double-digested restriction-associated DNA sequencing (ddRADseq) idea [ 10 – 12 ] has been along for some time, but its use for metagenome studies is quite new. The main advantage for this approach has been to reduce the sequencing efforts, and thereby costs per sample.…”

Reduced Metagenome Sequencing for strain-resolution taxonomic profiles

“…As an alternative to do a full shotgun metagenomic sequencing, the use of restriction enzymes to reduce the genomic sequence space has also been employed to investigate microbial communities [7][8][9]. The double-digested restriction associated DNA sequencing (ddRADseq) idea [10][11][12] has been along for some time, but its use for metagenome studies is quite new. The main advantage for this approach has been to reduce the sequencing efforts, and thereby costs per sample.…”