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Evaluating Hybridization Capture With RAD Probes as a Tool for Museum Genomics With Historical Bird Specimens

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Evaluating Hybridization Capture With RAD Probes as a Tool for Museum Genomics With Historical Bird Specimens

Ethan B Linck et al. Ecol Evol.

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.

Keywords: RADseq; ancient DNA; hyRAD; museum genomics; sequence capture.

Figures

Figure 1
Figure 1
Bioinformatics pipeline for S. torotoro hyRAD data. We demultiplexed 100‐bp paired‐end reads from three genomic libraries and filtered for adapter contamination/quality scores (A), or adapter contamination/quality scores and E. coli contamination (A1). Reads were clustered (as consensus fasta files) (B), and repeat regions removed from probes (C). After determining which assembled clusters were orthologous with probe regions (D), we merged flanking regions from on‐target loci in modern samples with the repeat‐free probe sequence to create a pseudo‐reference genome (E). To identify which contigs represented contamination in the original probe sample library from exogenous microbes or mitochondrial DNA, we BLAST searched against both the NCBI nt database and a full mitochondrial genome from S. torotoro relative Halcyon sanctus (F). We aligned quality filtered reads to this pseudo reference (G), called SNPs to produce a raw.vcf file for historic and modern DNA libraries separately (H). After filtering SNPs for origin in contaminant contigs and then restricting our matrix to sites present in both sample types (I), we filtered SNPs by read depth, quality scores, probability of being variable sites, and minor allele frequencies (J) prior to downstream analyses
Figure 2
Figure 2
Differences in sequencing and assembly performance between historical and modern DNA extractions. We observed significantly higher specificity (t = −17.711, df = 14.015, < .001), sensitivity (t = –12.928, df = 14.014, < .001), fold enrichment (t = −17.711, df = 14.015, < .001), and average coverage (t = −6.248, df = 7.555, < .001) in modern samples. We recovered a significantly higher total number of on‐target loci in modern samples (t = −12.239, df = 5.221, < .001), but significantly higher mean percent GC content in historic samples (t = 6.997, df = 13.368, < .001). We observed no significant differences between modern and historic samples for mean contig size or mean percentage of duplicate reads
Figure 3
Figure 3
Among historic samples, the number of trimmed reads was a significant predictor of the number of captured loci (R 2 = .872, < .001) and the initial sample DNA input quantity was a significant predictor of percent GC content in on‐target assembled contigs (R 2 = .370, = .016)
Figure 4
Figure 4
This displays the relationship between the cutoff for the minimum number of genotyped individuals required for a given SNP to be included in the data matrix and the total number of SNPs included in the resulting matrix
Figure 5
Figure 5
Individual membership probabilities for K = 3 ancestral populations inferred from analyzing 1,690 SNPs (100% complete data matrix) using discriminant analysis of principal components and retaining six principal component axes with two discriminant axes. Individual sampling locations are color coded accordingly

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References

    1. Andrews K. R., Good J. M., Miller M. R., Luikart G., & Hohenlohe P. A. (2016). Harnessing the power of RADseq for ecological and evolutionary genomics. Nature Reviews Genetics, 17(2), 81–92. - PMC - PubMed
    1. Axelsson E., Willerslev E., Gilbert M. T. P., & Nielsen R. (2008). The effect of ancient DNA damage on inferences of demographic histories. Molecular Biology and Evolution, 25, 2181–2187. - PubMed
    1. Baird N. A., Etter P. D., Atwood T. S., et al. (2008). Rapid SNP discovery and genetic mapping using sequenced RAD markers. PLoS One, 3, e3376. - PMC - PubMed
    1. Bankevich A., Nurk S., Antipov D., et al. (2012). SPAdes: A new genome assembly algorithm and its applications to single‐cell sequencing. Journal of Computational Biology, 19, 455–477. - PMC - PubMed
    1. Besnard G., Bertrand J. A. M., Delahaie B., et al. (2015). Valuing museum specimens: High‐throughput DNA sequencing on historical collections of New Guinea crowned pigeons (Goura). Biological Journal of the Linnean Society, 117, 71–82.

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