The Experts below are selected from a list of 26079 Experts worldwide ranked by ideXlab platform
Thomas M P Gilbert - One of the best experts on this subject based on the ideXlab platform.
-
characterizing restriction enzyme associated loci in historic ragweed ambrosia artemisiifolia voucher specimens using custom designed RNA Probes
Molecular Ecology Resources, 2017Co-Authors: Fatima Sanchez Barreiro, Filipe G Vieira, Michael D Martin, James Haile, Thomas M P Gilbert, Nathan WalesAbstract:Population genetic studies of non-model organisms frequently employ reduced representation library (RRL) methodologies, many of which rely on protocols in which genomic DNA is digested by one or more restriction enzymes. However, because high molecular weight DNA is recommended for these protocols, samples with degraded DNA are generally unsuitable for RRL methods. Given that ancient and historic specimens can provide key temporal perspectives to evolutionary questions, we explored how custom-designed RNA Probes could enrich for RRL loci (Restriction Enzyme-Associated Loci baits, or REALbaits). Starting with Genotyping-by-Sequencing (GBS) data generated on modern common ragweed (Ambrosia artemisiifolia L.) specimens, we designed 20,000 RNA Probes to target well-characterized genomic loci in herbarium voucher specimens dating from 1835–1913. Compared to shotgun sequencing, we observed enrichment of the targeted loci at 19–151-fold. Using our GBS capture pipeline on a dataset of 38 herbarium samples, we discovered 22,813 SNPs, providing sufficient genomic resolution to distinguish geographic populations. For these samples, we found that dilution of REALbaits to 10% of their original concentration still yielded sufficient data for downstream analyses and that a sequencing depth of ~7M reads was sufficient to characterize most loci without wasting sequencing capacity. In addition, we observed that targeted loci had highly variable rates of success, which we primarily attribute to similarity between loci, a trait that ultimately interferes with unambiguous read mapping. Our findings can help researchers design capture experiments for RRL loci, thereby providing an efficient means to integrate samples with degraded DNA into existing RRL datasets. This article is protected by copyright. All rights reserved.
Nathan Wales - One of the best experts on this subject based on the ideXlab platform.
-
characterizing restriction enzyme associated loci in historic ragweed ambrosia artemisiifolia voucher specimens using custom designed RNA Probes
Molecular Ecology Resources, 2017Co-Authors: Fatima Sanchez Barreiro, Filipe G Vieira, Michael D Martin, James Haile, Thomas M P Gilbert, Nathan WalesAbstract:Population genetic studies of non-model organisms frequently employ reduced representation library (RRL) methodologies, many of which rely on protocols in which genomic DNA is digested by one or more restriction enzymes. However, because high molecular weight DNA is recommended for these protocols, samples with degraded DNA are generally unsuitable for RRL methods. Given that ancient and historic specimens can provide key temporal perspectives to evolutionary questions, we explored how custom-designed RNA Probes could enrich for RRL loci (Restriction Enzyme-Associated Loci baits, or REALbaits). Starting with Genotyping-by-Sequencing (GBS) data generated on modern common ragweed (Ambrosia artemisiifolia L.) specimens, we designed 20,000 RNA Probes to target well-characterized genomic loci in herbarium voucher specimens dating from 1835–1913. Compared to shotgun sequencing, we observed enrichment of the targeted loci at 19–151-fold. Using our GBS capture pipeline on a dataset of 38 herbarium samples, we discovered 22,813 SNPs, providing sufficient genomic resolution to distinguish geographic populations. For these samples, we found that dilution of REALbaits to 10% of their original concentration still yielded sufficient data for downstream analyses and that a sequencing depth of ~7M reads was sufficient to characterize most loci without wasting sequencing capacity. In addition, we observed that targeted loci had highly variable rates of success, which we primarily attribute to similarity between loci, a trait that ultimately interferes with unambiguous read mapping. Our findings can help researchers design capture experiments for RRL loci, thereby providing an efficient means to integrate samples with degraded DNA into existing RRL datasets. This article is protected by copyright. All rights reserved.
Geoffrey B Fincher - One of the best experts on this subject based on the ideXlab platform.
-
low cost cross taxon enrichment of mitochondrial dna using in house synthesised RNA Probes
PLOS ONE, 2019Co-Authors: Stephen M Richards, Nelli Hovhannisyan, Matthew Gilliham, Joshua Ingram, Birgitte Skadhauge, Holly Heiniger, Bastien Llamas, Kieren J Mitchell, Julie Meachen, Geoffrey B FincherAbstract:Stephen M. Richards, Nelli Hovhannisyan, Matthew Gilliham, Joshua Ingram, Birgitte Skadhauge, Holly Heiniger, Bastien Llamas, Kieren J. Mitchell, Julie Meachen, Geoffrey B. Fincher, Jeremy J. Austin, Alan Cooper
-
Low-cost cross-taxon enrichment of mitochondrial DNA using in-house synthesised RNA Probes
2019Co-Authors: Stephen M Richards, Nelli Hovhannisyan, Matthew Gilliham, Joshua Ingram, Birgitte Skadhauge, Holly Heiniger, Bastien Llamas, Kieren J Mitchell, Julie Meachen, Geoffrey B FincherAbstract:Hybridization capture with in-solution oligonucleotide Probes has quickly become the preferred method for enriching specific DNA loci from degraded or ancient samples prior to high-throughput sequencing (HTS). Several companies synthesize sets of Probes for in-solution hybridization capture, but these commercial reagents are usually expensive. Methods for economical in-house probe synthesis have been described, but they do not directly address one of the major advantages of commercially synthesised Probes: that probe sequences matching many species can be synthesised in parallel and pooled. The ability to make “phylogenetically diverse” Probes increases the cost-effectiveness of commercial probe sets, as they can be used across multiple projects (or for projects involving multiple species). However, it is labour-intensive to replicate this with in-house methods, as template molecules must first be generated for each species of interest. While it has been observed that Probes can be used to enrich for phylogenetically distant targets, the ability of this effect to compensate for the lack of phylogenetically diverse Probes in in-house synthesised probe sets has not been tested. In this study, we present a refined protocol for in-house RNA probe synthesis and evaluated the ability of Probes generated using this method from a single species to successfully enrich for the target locus in phylogenetically distant species. We demonstrated that Probes synthesized using long-range PCR products from a placental mammal mitochondrion (Bison spp.) could be used to enrich for mitochondrial DNA in birds and marsupials (but not plants). Importantly, our results were obtained for approximately a third of the cost of similar commercially available reagents.
James Haile - One of the best experts on this subject based on the ideXlab platform.
-
characterizing restriction enzyme associated loci in historic ragweed ambrosia artemisiifolia voucher specimens using custom designed RNA Probes
Molecular Ecology Resources, 2017Co-Authors: Fatima Sanchez Barreiro, Filipe G Vieira, Michael D Martin, James Haile, Thomas M P Gilbert, Nathan WalesAbstract:Population genetic studies of non-model organisms frequently employ reduced representation library (RRL) methodologies, many of which rely on protocols in which genomic DNA is digested by one or more restriction enzymes. However, because high molecular weight DNA is recommended for these protocols, samples with degraded DNA are generally unsuitable for RRL methods. Given that ancient and historic specimens can provide key temporal perspectives to evolutionary questions, we explored how custom-designed RNA Probes could enrich for RRL loci (Restriction Enzyme-Associated Loci baits, or REALbaits). Starting with Genotyping-by-Sequencing (GBS) data generated on modern common ragweed (Ambrosia artemisiifolia L.) specimens, we designed 20,000 RNA Probes to target well-characterized genomic loci in herbarium voucher specimens dating from 1835–1913. Compared to shotgun sequencing, we observed enrichment of the targeted loci at 19–151-fold. Using our GBS capture pipeline on a dataset of 38 herbarium samples, we discovered 22,813 SNPs, providing sufficient genomic resolution to distinguish geographic populations. For these samples, we found that dilution of REALbaits to 10% of their original concentration still yielded sufficient data for downstream analyses and that a sequencing depth of ~7M reads was sufficient to characterize most loci without wasting sequencing capacity. In addition, we observed that targeted loci had highly variable rates of success, which we primarily attribute to similarity between loci, a trait that ultimately interferes with unambiguous read mapping. Our findings can help researchers design capture experiments for RRL loci, thereby providing an efficient means to integrate samples with degraded DNA into existing RRL datasets. This article is protected by copyright. All rights reserved.
Michael D Martin - One of the best experts on this subject based on the ideXlab platform.
-
characterizing restriction enzyme associated loci in historic ragweed ambrosia artemisiifolia voucher specimens using custom designed RNA Probes
Molecular Ecology Resources, 2017Co-Authors: Fatima Sanchez Barreiro, Filipe G Vieira, Michael D Martin, James Haile, Thomas M P Gilbert, Nathan WalesAbstract:Population genetic studies of non-model organisms frequently employ reduced representation library (RRL) methodologies, many of which rely on protocols in which genomic DNA is digested by one or more restriction enzymes. However, because high molecular weight DNA is recommended for these protocols, samples with degraded DNA are generally unsuitable for RRL methods. Given that ancient and historic specimens can provide key temporal perspectives to evolutionary questions, we explored how custom-designed RNA Probes could enrich for RRL loci (Restriction Enzyme-Associated Loci baits, or REALbaits). Starting with Genotyping-by-Sequencing (GBS) data generated on modern common ragweed (Ambrosia artemisiifolia L.) specimens, we designed 20,000 RNA Probes to target well-characterized genomic loci in herbarium voucher specimens dating from 1835–1913. Compared to shotgun sequencing, we observed enrichment of the targeted loci at 19–151-fold. Using our GBS capture pipeline on a dataset of 38 herbarium samples, we discovered 22,813 SNPs, providing sufficient genomic resolution to distinguish geographic populations. For these samples, we found that dilution of REALbaits to 10% of their original concentration still yielded sufficient data for downstream analyses and that a sequencing depth of ~7M reads was sufficient to characterize most loci without wasting sequencing capacity. In addition, we observed that targeted loci had highly variable rates of success, which we primarily attribute to similarity between loci, a trait that ultimately interferes with unambiguous read mapping. Our findings can help researchers design capture experiments for RRL loci, thereby providing an efficient means to integrate samples with degraded DNA into existing RRL datasets. This article is protected by copyright. All rights reserved.
