Authors: Shi Wang, Eli Meyer, John McKay & Mikhail Matz
High-throughput sequencing of restriction site-associated genomic DNA (RAD) is now a widely used approach for simultaneously discovering and profiling genetic polymorphisms. We describe an alternative approach for RAD genotyping with several useful features, called 2b-RAD for the use of type IIB restriction endonucleases (AlfI, BsaXI). The fragments excised by type IIB enzymes are uniform in length, streamlining library preparation and producing even sequencing coverage across the genome. The cohesive ends present on both 5’ and 3’ ends of each fragment make it possible to target a defined subset of restriction sites using modified adaptors. Marker density can be adjusted as needed in this way, greatly reducing sequencing coverage requirements and costs. 2b-RAD offers a cost-effective strategy for high-throughput genotyping that is widely suitable for linkage mapping and profiling genetic variation in natural populations.
Sample preparation for 2b-RAD genotyping is accomplished in a single well of a multi-well plate within about 4 hours. 2b-RAD constructs are produced in a series of three reactions (restriction digest, adaptor ligation, and barcode incorporation), then gel-purified and sequenced (Fig 1).
Figure 1: Preparation of samples for 2b-RAD genotyping.
Sample preparation for 2b-RAD genotyping is accomplished by restriction digest (AlfI) of genomic DNA, cohesive end ligation of partially double-stranded adaptors with compatible (NN) overhangs, and incorporation of barcodes for multiplex sequencing by PCR.
First, genomic DNA is digested using type IIB restriction endonucleases to excise the target fragments used for genotyping (Fig 1). To maintain buffer concentrations in subsequent reactions, the initial digest is conducted in a small volume, which necessitates high DNA concentrations (at least 250 ng µl-1). Samples with lower concentrations can be easily concentrated using standard methods including ethanol precipitation or drying under vacuum.
Before processing a new set of samples (e.g. a new species, etc.), it’s advisable to test the effectiveness of the chosen restriction enzyme by comparing digested genomic DNA (~100-200 ng genomic DNA) against the uncut DNA and a no-enzyme control. While partially degraded DNA can be used effectively for 2b-RAD genotyping, for the purposes of this test a perfectly intact sample should be chosen. Digested samples show a subtle smear extending downward from the original high-molecular weight genomic DNA band. No-enzyme controls should look identical to the original DNA after 1 hr at 37°C.
Next, partially double-stranded adaptors are ligated to the restriction fragments using T4 DNA ligase. The sequences of these adaptors, and all other oligonucleotides used in this protocol, are shown in Table 1. Note that the choice of adaptors at this stage determines the set of restriction sites to be included in the library, and the sequencing platform to be used. The fully degenerate overhangs (5’-NNN-3’) featured in the standard protocol target all sites. Adaptors with reduced degeneracy can be used instead to target a subset of the sites (e.g. adaptors bearing overhangs 5’-NNG-3’ target 1/16th of the sites). The fraction targeted can be chosen based on genome size, polymorphism frequency, and the number of polymorphisms required for a particular experiment.
Table 1: Oligonucleotide sequences for 2b-RAD sample preparation
bc: barcode, any 6-10 bp DNA sequence easily distinguishable from other barcodes on that same sequencing run.
In the final reaction, ligation products are amplified using oligonucleotide primers complementary to the adaptors, and incorporating sample-specific barcodes for multiplex sequencing of pooled samples. This is accomplished using a step-out PCR procedure in which two long oligos present at low concentrations incorporate barcode sequences and binding sites for two shorter primers present at standard concentrations. To minimize any distortions of allele frequencies, samples are collected across a range of cycle numbers to identify the minimum number of cycles required to produce a visible product in gel electrophoresis. Only a small number of cycles should be required (4-10) for standard libraries, while for reduced schemes additional cycles proportional to the targets’ lower abundance are required (e.g. 1/16th reductions require an additional ~4 cycles).
Finally, the barcoded PCR products are gel purified using standard methods to remove primer dimers and residual products of restriction digest. The purified products are then pooled for multiplex sequencing on either SOLiD or Illumina platforms. Note that the fragments produced by 2b-RAD are shorter than the maximum read length of these technologies, so short read lengths should be specified when submitting samples (36 bp or less) to minimize sequencing costs.
The details given in Procedures correspond to a standard AlfI library to be sequenced on the SOLiD System. A wide variety of modifications are possible, depending on the choice of enzyme, the reduction scheme, and the sequencing platform, as described in the associated manuscript.
2b-RAD: a simple and flexible method for genome-wide genotyping. Shi Wang, Eli Meyer, John K McKay, and Mikhail V Matz. Nature Methods doi:10.1038/nmeth.2023
Shi Wang, College of Marine Life Sciences, Ocean University of China
Eli Meyer, Department of Zoology, Oregon State
John McKay, Department of Bioagricultural Sciences and Graduate Degree Program in Ecology, Colorado State University
Mikhail Matz, School of Biological Sciences, University of Texas - Austin
Source: Protocol Exchange (2012) doi:10.1038/protex.2012.010. Originally published online 6 June 2012.