Targeted enrichment strategies for next-generation plant biology.
OBJECTIVE
The dramatic advances offered by modern DNA sequencers continue to redefine the limits of what can be accomplished in comparative plant biology. Even with recent achievements, however, plant genomes present obstacles that can make it difficult to execute large-scale population and phylogenetic studies on next-generation sequencing platforms. Factors like large genome size, extensive variation in the proportion of organellar DNA in total DNA, polyploidy, and gene number/redundancy contribute to these challenges, and they demand flexible targeted enrichment strategies to achieve the desired goals.


METHODS
In this article, we summarize the many available targeted enrichment strategies that can be used to target partial-to-complete organellar genomes, as well as known and anonymous nuclear targets. These methods fall under four categories: PCR-based enrichment, hybridization-based enrichment, restriction enzyme-based enrichment, and enrichment of expressed gene sequences.


RESULTS
Examples of plant-specific applications exist for nearly all methods described. While some methods are well established (e.g., transcriptome sequencing), other promising methods are in their infancy (hybridization enrichment). A direct comparison of methods shows that PCR-based enrichment may be a reasonable strategy for accessing small genomic targets (e.g., ≤50 kbp), but that hybridization and transcriptome sequencing scale more efficiently if larger targets are desired.


CONCLUSIONS
While the benefits of targeted sequencing are greatest in plants with large genomes, nearly all comparative projects can benefit from the improved throughput offered by targeted multiplex DNA sequencing, particularly as the amount of data produced from a single instrument approaches a trillion bases per run.
Pacific Northwest Research Station, USDA Forest Service, Corvallis, Oregon 97331, USA. rcronn@fs.fed.us
Richard
R
Cronn
Brian J
BJ
Knaus
Aaron
A
Liston
Peter J
PJ
Maughan
Matthew
M
Parks
John V
JV
Syring
Joshua
J
Udall
D001483:Base Sequence; D002467:Cell Nucleus / Q000235:genetics; D002736:Chloroplasts / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D020869:Gene Expression Profiling; D054628:Genome, Chloroplast; D018745:Genome, Plant; D060005:Genotyping Techniques / Q000379:methods; D010802:Phylogeny; D010944:Plants / Q000145:classification / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000379:methods*
D018744:DNA, Plant