RAD paired-end sequencing for local de novo assembly and SNP discovery in non-model organisms

Methods Mol Biol. 2012:888:135-51. doi: 10.1007/978-1-61779-870-2_9.

Abstract

Restriction-site Associated DNA (RAD) markers are rapidly becoming a standard for SNP discovery and genotyping studies even in organisms without a sequenced reference genome. It is difficult, however, to identify genes nearby RAD markers of interest or move from SNPs identified by RAD to a high-throughput genotyping assay. Paired-end sequencing of RAD fragments can alleviate these problems by generating a set of paired sequences that can be locally assembled into high-quality contigs up to 1 kb in length. These contigs can then be used for SNP identification, homology searching, or high-throughput assay primer design. In this chapter, we offer suggestions on how to design a RAD paired-end (RAD-PE) sequencing project and the protocol for creating paired-end RAD libraries suitable for Illumina sequencers.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Contig Mapping
  • DNA Primers / genetics
  • Gene Library
  • Genetic Markers / genetics
  • Genome*
  • Genotype
  • High-Throughput Nucleotide Sequencing / methods*
  • Polymorphism, Single Nucleotide / genetics*
  • Sequence Analysis, DNA / methods*
  • Smegmamorpha / genetics*

Substances

  • DNA Primers
  • Genetic Markers