Abstract
Laboratory techniques for high‐throughput sequencing have enhanced our ability to generate DNA sequence data from millions of natural history specimens collected prior to the molecular era, but remain poorly tested at shallower evolutionary time scales. Hybridization capture using restriction site‐associated DNA probes (hyRAD) is a recently developed method for population genomics with museum specimens. The hyRAD method employs fragments produced in a restriction site‐associated double digestion as the basis for probes that capture orthologous loci in samples of interest. While promising in that it does not require a reference genome, hyRAD has yet to be applied across study systems in independent laboratories. Here, we provide an independent assessment of the effectiveness of hyRAD on both fresh avian tissue and dried tissue from museum specimens up to 140 years old and investigate how variable quantities of input DNA affect sequencing, assembly, and population genetic inference. We present a modified bench protocol and bioinformatics pipeline, including three steps for detection and removal of microbial and mitochondrial DNA contaminants. We confirm that hyRAD is an effective tool for sampling thousands of orthologous SNPs from historic museum specimens to describe phylogeographic patterns. We find that modern DNA performs significantly better than historical DNA better during sequencing but that assembly performance is largely equivalent. We also find that the quantity of input DNA predicts %GC content of assembled contiguous sequences, suggesting PCR bias. We caution against sampling schemes that include taxonomic or geographic autocorrelation across modern and historic samples.
Highlights
Over the past three decades, novel laboratory techniques have enhanced our ability to generate DNA sequence data from millions of natural history specimens collected prior to the molecular era (Payne & Sorenson 2002)
The majority of reads lost to quality control were PCR duplicates, with a range of 50.9% to 91.1% duplicate reads per sample. 2,455 reads were removed as E. coli contamination from 12 of 20 individuals (Table 2)
Hybridization-restriction site associated DNA (RAD) is an effective tool for sampling thousands of orthologous SNPs from historic museum specimens
Summary
Over the past three decades, novel laboratory techniques have enhanced our ability to generate DNA sequence data from millions of natural history specimens collected prior to the molecular era (Payne & Sorenson 2002). High-throughput sequencing has dramatically increased both the overall efficiency of data collection and the total amount of sequence data that it is possible to collect from museum specimens (Rizzi et al 2012; Hofreiter et al 2015) by overcoming scalability hurdles intrinsic to traditional Sanger sequencing methods (Soltis & Soltis 1993; Wandeler et al 2007). High-throughput sequencing has already proved widely useful for incorporating museum specimens into phylogenomic studies (Burbano et al 2010; McCormack et al 2012; Besnard et al 2015), its application for collecting genome-wide markers at the population level has lagged behind its use for addressing questions at deeper evolutionary time scales due to limitations in the most commonly employed library preparation methods for reducedrepresentation Illumina sequencing (Suchan et al 2016). Our understanding of the efficacy and biases of different approaches to reduced-representation genome sequencing from degraded DNA remains incomplete relative to either Sanger sequencing (Soltis & Soltis 1993; Wandeler et al 2007) or high-coverage, single-sample whole genome sequencing (Poinar et al 2006)
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