TY - JOUR

T1 - Characterizing restriction enzyme-associated loci in historic ragweed (Ambrosia artemisiifolia) voucher specimens using custom-designed RNA probes

AU - Sanchez Barreiro, Fatima

AU - Garrett Vieira, Filipe Jorge

AU - Martin, Michael David

AU - Haile, James Seymour

AU - Gilbert, Tom

AU - Wales, Nathan

N1 - This article is protected by copyright. All rights reserved.

PY - 2017

Y1 - 2017

N2 - 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.

AB - 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.

U2 - 10.1111/1755-0998.12610

DO - 10.1111/1755-0998.12610

M3 - Journal article

C2 - 27762072

VL - 17

SP - 209

EP - 220

JO - Molecular Ecology

JF - Molecular Ecology

SN - 0962-1083

IS - 2

ER -