RAD Capture (Rapture): Flexible and Efficient Sequence-Based Genotyping

Abstract

Massively parallel sequencing has revolutionized many areas of biology, but sequencing large amounts of DNA in many individuals is cost-prohibitive and unnecessary for many studies. Genomic complexity reduction techniques such as sequence capture and restriction enzyme-based methods enable the analysis of many more individuals per unit cost. Despite their utility, current complexity reduction methods have limitations, especially when large numbers of individuals are analyzed. Here we develop a much improved restriction site-associated DNA (RAD) sequencing protocol and a new method called Rapture ( R: AD c APTURE: ). The new RAD protocol improves versatility by separating RAD tag isolation and sequencing library preparation into two distinct steps. This protocol also recovers more unique (nonclonal) RAD fragments, which improves both standard RAD and Rapture analysis. Rapture then uses an in-solution capture of chosen RAD tags to target sequencing reads to desired loci. Rapture combines the benefits of both RAD and sequence capture, i.e., very inexpensive and rapid library preparation for many individuals as well as high specificity in the number and location of genomic loci analyzed. Our results demonstrate that Rapture is a rapid and flexible technology capable of analyzing a very large number of individuals with minimal sequencing and library preparation cost. The methods presented here should improve the efficiency of genetic analysis for many aspects of agricultural, environmental, and biomedical science.

Keywords: genotyping; massively parallel sequencing; population genetics; rainbow trout; restriction-site associated DNA (RAD); sequence capture.

Figure 1

Schematic overview of the new RAD protocol and RAD capture (Rapture) method. (A) RAD tag isolation. Two wells are depicted in each of two different plates. Genomic DNA is digested with a restriction enzyme and ligated to biotinylated well barcode adaptors (yellow and blue bars). (B) RAD tag isolation and library preparation. DNA from each well is pooled platewise, mechanically sheared, and incubated with streptavidin beads. Following washing, DNA is cleaved from the beads leaving the well barcodes. Finally, a library preparation is performed where a unique plate barcode is added (red and purple bars). (C) Rapture. Multiple plate libraries are pooled, hybridized to biotinylated oligonucleotide baits corresponding to the targeted RAD tag loci, and captured to produce the final library enriched for the loci of interest.

Figure 2

Comparison of RAD sequencing results from traditional and new RAD protocols on 96 individuals. (A) Histogram showing the number of individuals per bin of mapped fragments without clone removal. (B) Scatter plot showing the relationship between the number of loci covered ≥4× without clone removal and the number of sequenced fragments per individual. (C) Histogram showing the number of individuals per bin of mapped fragments with clone removal. (D) Scatter plot showing the relationship between the number loci covered ≥4× with clone removal and the number of sequenced fragments per individual.

Figure 3

Comparisons of RAD, Rapture, and Rapture with one-fifth bait concentration (Rapture 1/5) sequencing results with clone removal on 288 individuals. (A) Histogram showing the number of individuals per bin of mapped fragments. (B) Scatter plot showing the relationship between number of Rapture loci covered ≥4× and the number of sequenced fragments per individual. (C) Scatter plot showing the relationship between the number of non-Rapture loci covered ≥4× and the number of sequenced fragments per individual. (D) Scatter plot showing the relationship between number Rapture loci covered at select levels and the number of sequenced fragments per individual for Rapture 1/5. (E) Histogram showing the number of Rapture loci covered ≥4× per bin individual for Rapture 1/5. (F) Histogram showing number of non-Rapture loci covered ≥4× per bin individual for Rapture 1/5.

Figure 4

SNP discovery using Rapture with one-fifth bait concentration data. (A) Histogram showing the number of SNPs per bin of position in Rapture locus. (B–D) Scatter plots showing the relationship between the number of individuals genotyped and SNP position using different posterior probability cutoffs. (E) Scatter plot showing the relationship between the number of SNPs genotyped and the number of sequenced fragments per individual for SNPs in position 1–84. (F) Scatter plot showing the relationship between the number of SNPs genotyped and number of sequenced fragments per individual for SNPs in position 85–500.

Figure 5

Principal component analysis of Rapture genotyping results from Fall River rainbow trout. Individuals are labeled based on birth location. Individuals with known birth locations were collected as juveniles near spawning grounds. Other individuals were collected as adults throughout the system below the spawning grounds. (A) Scatter plot showing the first two principal components. (B) Scatter plot showing the first and third principal components. (C) Fall River map.