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Abstract Next‐generation sequencing technologies now allow researchers of non‐model systems to perform genome‐based studies without the requirement of a (often unavailable) closely related genomic reference. We evaluated the role of restriction endonuclease (RE) selection in double‐digest restriction‐site‐associatedDNAsequencing (ddRADseq) by generating reduced representation genome‐wide data using four differentREcombinations. Our expectation was thatREselections targeting longer, more complex restriction sites would recover fewer loci thanREwith shorter, less complex sites. We sequenced a diverse sample of non‐model arachnids, including five congeneric pairs of harvestmen (Opiliones) and four pairs of spiders (Araneae). Sample pairs consisted of either conspecifics or closely related congeneric taxa, and in total 26 sample pair analyses were tested. Sequence demultiplexing, read clustering and variant calling were performed in thepyRADprogram. The 6‐base pair cutterEcoRIcombined with methylated site‐specific 4‐base pair cutterMspIproduced, on average, the greatest numbers of intra‐individual loci and shared loci per sample pair. As expected, the number of shared loci recovered for a sample pair covaried with the degree of genetic divergence, estimated with cytochrome oxidase I sequences, although this relationship was non‐linear. Our comparative results will prove useful in guiding protocol selection for ddRADseq experiments on many arachnid taxa where reference genomes, even from closely related species, are unavailable.
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A basic dd RAD seq two‐enzyme protocol performs well with herbarium and silica‐dried tissues across four genera
PremiseThe ability to sequence genome‐scale data from herbarium specimens would allow for the economical development of data sets with broad taxonomic and geographic sampling that would otherwise not be possible. Here, we evaluate the utility of a basic double‐digest restriction site–associatedDNAsequencing (ddRADseq) protocol usingDNAs from four genera extracted from both silica‐dried and herbarium tissue. MethodsDNAs fromDraba,Boechera,Solidago, andIlexwere processed with a ddRADseq protocol. The effects ofDNAdegradation, taxon, and specimen age were assessed. ResultsAlthough taxon, preservation method, and specimen age affected data recovery, large phylogenetically informative data sets were obtained from the majority of samples. DiscussionThese results suggest that herbarium samples can be incorporated into ddRADseq project designs, and that specimen age can be used as a rapid on‐site guide for sample choice. The detailed protocol we provide will allow users to pursue herbarium‐based ddRADseq projects that minimize the expenses associated with fieldwork and sample evaluation.
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- Award ID(s):
- 1920858
- PAR ID:
- 10455777
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Applications in Plant Sciences
- Volume:
- 8
- Issue:
- 4
- ISSN:
- 2168-0450
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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