double restriction enzyme re digest  (New England Biolabs)

Journal: PLoS ONE

Article Title: Double Digest RADseq: An Inexpensive Method for De Novo SNP Discovery and Genotyping in Model and Non-Model Species

doi: 10.1371/journal.pone.0037135

Figure Lengend Snippet: ( A ) Traditional Restriction-Site Associated DNA sequencing (RADseq) uses a single restriction enzyme (RE) digest coupled with secondary random fragmentation and broad size selection to generate reduced representation libraries consisting of all genomic regions adjacent to the RE cut site (red segments). ( B ) Double digest RAD sequencing (ddRADseq), by contrast, uses a two enzyme double digest followed by precise size selection that excludes regions flanked by either [a] very close or [b] very distant RE recognition sites, recovering a library consisting of only fragments close to the target size (red segments). Representation in this library is expected to be inversely proportional to deviation from the size-selection target, thus read counts across regions are expected to be correlated between individuals (yellow and green bars).

Article Snippet: Instead, we use a double restriction enzyme (RE) digest (i.e., a restriction digest with two enzymes simultaneously) that results in at least five-fold reduction in library production cost–complete ddRADseq libraries cost ∼$5 per sample, while the necessary enzymatic steps following the initial restriction digest and ligation in random shearing RAD libraries alone introduce a cost of ∼$25 per library (NEB, Ipswich, MA).

Techniques: DNA Sequencing, Selection, Sequencing

Journal: PLoS ONE

Article Title: Double Digest RADseq: An Inexpensive Method for De Novo SNP Discovery and Genotyping in Model and Non-Model Species

doi: 10.1371/journal.pone.0037135

Figure Lengend Snippet: Changing the restriction enzyme (RE) or size-selection regime modifies the fraction of genome recovered. Simulation 1 (blue lines, shading): the expected fragment size distribution for a RE digest with NlaIII and MluCI (CATG and AATT) in the Mus musculus genome is shown (solid blue line). “Broad” size selection (300 bp±50 bp) is modeled by a normal sampling distribution (mean = 300 bp, SD = 25 bp). Under this sampling distribution, 4,900,000 sequence reads (dashed blue line) are expected to cover ∼119,000 regions at 7× or greater (blue area). Simulation 2 (red lines, shading): the expected fragment size distribution for a digest with EcoRI and MspI (GAATTC and CCGG) is shown (solid red line). “Narrow” size selection (300 bp±24 bp; see text) is modeled by a normal sampling distribution (mean = 300 bp, SD = 11 bp; see Supporting ). Under this sampling distribution, an investment of 315,000 sequence reads (dashed red line) is sufficient to recover ∼17,000 regions at 7× or greater (red area).

Article Snippet: Instead, we use a double restriction enzyme (RE) digest (i.e., a restriction digest with two enzymes simultaneously) that results in at least five-fold reduction in library production cost–complete ddRADseq libraries cost ∼$5 per sample, while the necessary enzymatic steps following the initial restriction digest and ligation in random shearing RAD libraries alone introduce a cost of ∼$25 per library (NEB, Ipswich, MA).

Techniques: Selection, Sampling, Sequencing