An official website of the United States government
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.
more »
« less
Expressed exome capture sequencing: A method for cost‐effective exome sequencing for all organisms
Abstract Exome capture is an effective tool for surveying the genome for loci under selection. However, traditional methods require annotated genomic resources. Here, we present a method for creatingcDNAprobes from expressedmRNA, which are then used to enrich and capture genomicDNAfor exon regions. This approach, called “EecSeq,” eliminates the need for costly probe design and synthesis. We tested EecSeq in the eastern oyster,Crassostrea virginica, using a controlled exposure experiment. Four adult oysters were heat shocked at 36°C for 1 hr along with four control oysters kept at 14°C. StrandedmRNAlibraries were prepared for two individuals from each treatment and pooled. Half of the combined library was used for probe synthesis, and half was sequenced to evaluate capture efficiency. GenomicDNAwas extracted from all individuals, enriched via captured probes, and sequenced directly. We found that EecSeq had an average capture sensitivity of 86.8% across all known exons and had over 99.4% sensitivity for exons with detectable levels of expression in themRNAlibrary. For all mapped reads, over 47.9% mapped to exons and 37.0% mapped to expressed targets, which is similar to previously published exon capture studies. EecSeq displayed relatively even coverage within exons (i.e., minor “edge effects”) and even coverage across exonGCcontent. We discovered 5,951SNPs with a minimum average coverage of 80×, with 3,508SNPs appearing in exonic regions. We show that EecSeq provides comparable, if not superior, specificity and capture efficiency compared to costly, traditional methods.
more »
« less
- Award ID(s):
- 1635423
- PAR ID:
- 10060536
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- Molecular Ecology Resources
- Volume:
- 18
- Issue:
- 6
- ISSN:
- 1755-098X
- Page Range / eLocation ID:
- p. 1209-1222
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Abstract The development of high‐throughput sequencing technologies is dramatically increasing the use of single nucleotide polymorphisms (SNPs) across the field of genetics, but most parentage studies of wild populations still rely on microsatellites. We developed a bioinformatic pipeline for identifyingSNPpanels that are informative for parentage analysis from restriction site‐associatedDNAsequencing (RADseq) data. This pipeline includes options for analysis with or without a reference genome, and provides methods to maximize genotyping accuracy and select sets of unlinked loci that have high statistical power. We test this pipeline on small populations of Mexican gray wolf and bighorn sheep, for which parentage analyses are expected to be challenging due to low genetic diversity and the presence of many closely related individuals. We compare the results of parentage analysis acrossSNPpanels generated with or without the use of a reference genome, and betweenSNPs and microsatellites. For Mexican gray wolf, we conducted parentage analyses for 30 pups from a single cohort where samples were available from 64% of possible mothers and 53% of possible fathers, and the accuracy of parentage assignments could be estimated because true identities of parents were known a priori based on field data. For bighorn sheep, we conducted maternity analyses for 39 lambs from five cohorts where 77% of possible mothers were sampled, but true identities of parents were unknown. Analyses with and without a reference genome producedSNPpanels with ≥95% parentage assignment accuracy for Mexican gray wolf, outperforming microsatellites at 78% accuracy. Maternity assignments were completely consistent across allSNPpanels for the bighorn sheep, and were 74.4% consistent with assignments from microsatellites. Accuracy and consistency of parentage analysis were not reduced when using as few as 284SNPs for Mexican gray wolf and 142SNPs for bighorn sheep, indicating our pipeline can be used to developSNPgenotyping assays for parentage analysis with relatively small numbers of loci.more » « less
-
Abstract Developing improved fluorescent probes for imaging the endoplasmic reticulum (ER) is necessary for structure‐activity studies of this dynamic organelle. Two coumarin‐based compounds with sulfonamide side groups were synthesized and characterized asER‐targeting probes. Their selectivity to target theERin HeLa andGM07373 mammalian cells was shown with co‐localization experiments using commercially available probes that localize in theER, mitochondria, or lysozymes. The hydrophobicity of the coumarin‐based probes was comparable to known probes that partition into theERmembrane. Their cytotoxicity in mammalian cells was low withIC50 values that range from 205 to 252 μm. The fluorescent quantum yields of the coumarin‐based probes when excited with 400 nm light were 0.60, and they have a much narrower emission spectrum (from 435 to 525 nm in methanol) than that of the only commercially availableERprobe that is exited with 400 nm light (ER‐Tracker™ Blue‐WhiteDPX). Thus, the coumarin‐based probes are more useful for multicolor imaging with yellow and red emitting fluorophores. In addition to the above benefits,ERlabeling was achieved with the coumarin‐based probes in both live cells and fixed cells, revealing their versatility for a wide range of cellular imaging applications.more » « less
-
Abstract Single‐nucleotide polymorphisms (SNPs) are preferred over microsatellite markers in many evolutionary studies, but have only recently been applied to studies of parentage. Evaluations ofSNPs and microsatellites for assigning parentage have mostly focused on special cases that require a relatively large number of heterozygous loci, such as species with low genetic diversity or with complex social structures. We developed 120SNPmarkers from a transcriptome assembled usingRNA‐sequencing of a songbird with the most common avian mating system—social monogamy. We compared the effectiveness of 97 novelSNPs and six previously described microsatellites for assigning paternity in the black‐throated blue warbler,Setophaga caerulescens. We show that the full panel of 97SNPs (meanHo = 0.19) was as powerful for assigning paternity as the panel of multiallelic microsatellites (meanHo = 0.86). Paternity assignments using the two marker types were in agreement for 92% of the offspring. Filtering individual samples by a 50% call rate andSNPs by a 75% call rate maximized the number of offspring assigned with 95% confidence usingSNPs. We also found that the 40 most heterozygousSNPs (meanHo = 0.37) had similar power to assign paternity as the full panel of 97SNPs. These findings demonstrate that a relatively small number of variableSNPs can be effective for parentage analyses in a socially monogamous species. We suggest that the development ofSNPmarkers is advantageous for studies that require high‐throughput genotyping or that plan to address a range of ecological and evolutionary questions.more » « less
-
ABSTRACT Coral reefs are increasingly threatened by disease outbreaks, yet little is known about the genetic mechanisms underlying disease resistance. Since the 1970s, White Band Disease (WBD) has decimated the Caribbean staghorn coralAcropora cervicornis. However, 15% or more of individuals are highly disease‐resistant, and the genes controlling the production of Argonaut proteins, involved in microRNA (miRNA) post‐transcriptional gene silencing, are up‐regulated in WBD‐resistant corals. This suggests that miRNAs may be key regulators of coral immunity. In this study, we conducted an in situ disease transmission experiment with five healthy‐exposed control tanks and five WBD‐exposed tanks, each containing 50A. cervicornisgenotypes, sampled over 7 days and then sequenced miRNAs from 12 replicate genotypes, including 12 WBD‐exposed and 12 healthy‐exposed control fragments from two time points. We identified 67bona fidemiRNAs inA. cervicornis, 3 of which are differentially expressed in disease‐resistant corals. We performed a phylogenetic comparison of miRNAs across cnidarians and found greater conservation of miRNAs in more closely related taxa, including all three differentially expressed miRNAs being conserved in more than oneAcroporacoral. One of the three miRNAs has putative genomic targets involved in the cnidarian innate immunity. In addition, community detection coupled with over‐representation analysis of our miRNA–messenger RNA (mRNA) target network found two key uniqueA. cervicornismiRNAs regulating multiple important immune‐related pathways such as Toll‐like receptor pathway, endocytosis, and apoptosis. These findings highlight how multiple miRNAs may help the coral host maintain immune homeostasis in the presence of environmental stress including disease.more » « less
