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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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The problem of genetic code misreading during protein synthesis
Abstract Saccharomyces cerevisiaehas been an important model for determining the frequency of translational misreading events, those in which a tRNA pairs incorrectly to the mRNA and inserts an amino acid not specified by the codon in the mRNA. Misreading errors have been quantified in vivo using reporter protein systems or mass spectrometry with both approaches converging on a simple model for most misreading. The available data show that misreading tRNAs must form stereotypical base mismatches that correspond to those that can mimic Watson–Crick base pairs when formed in the ribosomal A site. Errors involving other mismatches occur significantly less frequently. This work debunks the idea of an average misreading frequency of 5 × 10−4per codon that extends across the genetic code. Instead, errors come in two distinct classes—high frequency and low frequency events—with most errors being of the low frequency type. A comparison of misreading errors inS. cerevisiaeandEscherichia colisuggests the existence of a mechanism that reduces misreading frequency in yeast; this mechanism may operate in eukaryotes generally.
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- Award ID(s):
- 1645795
- PAR ID:
- 10462183
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Yeast
- Volume:
- 36
- Issue:
- 1
- ISSN:
- 0749-503X
- Page Range / eLocation ID:
- p. 35-42
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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