Multiple-complete-digest restriction fragment mapping: Generating sequence-ready maps for large-scale DNA sequencing

Abstract

Multiple-complete-digest mapping is a DNA mapping technique based on complete-restriction-digest fingerprints of a set of clones that provides highly redundant coverage of the mapping target. The maps assembled from these fingerprints order both the clones and the restriction fragments. Maps are coordinated across three enzymes in the examples presented. Starting with yeast artificial chromosome contigs from the 7q31.3 and 7p14 regions of the human genome, we have produced cosmid-based maps spanning more than one million base pairs. Each yeast artificial chromosome is first subcloned into cosmids at a redundancy of ؋15-30. Complete-digest fragments are electrophoresed on agarose gels, poststained, and imaged on a f luorescent scanner. Aberrant clones that are not representative of the underlying genome are rejected in the map construction process. Almost every restriction fragment is ordered, allowing selection of minimal tiling paths with clone-to-clone overlaps of only a few thousand base pairs. These maps demonstrate the practicality of applying the experimental and software-based steps in multiple-complete-digest mapping to a target of significant size and complexity. We present evidence that the maps are sufficiently accurate to validate both the clones selected for sequencing and the sequence assemblies obtained once these clones have been sequenced by a ''shotgun'' method.

Sequence Ready—or Not?

“…1). An alternate approach is to subclone YAC clones into cosmids directly (Wong et al 1997). Once the appropriate clones have been identified, the next step is to assemble these clones into an ordered contig.…”

“…Once the appropriate clones have been identified, the next step is to assemble these clones into an ordered contig. Clones are subjected to restriction endonuclease digestion, and fragments are separated by gel electrophoresis (Marra et al 1997;Wong et al 1997). Overlapping clones are then identified by shared restriction pattern or ''fingerprint'' similarities (see Fig.…”

Vertically integrated analysis of human DNA. Final technical report

“…Note: A manuscript published subsequent to the completion of this project (Wong et al, 1997) provides detailed documentation of the success of large-scale applications of cosmid-level MCD mapping. Most of the R&D behind this first large-scale test of the system was supported under DE-FG06-92ER61487.…”

High Throughput Fingerprint Analysis of Large-Insert Clones

“…Similar to studies by Olson et al (1986) and Wong et al (1997), data from restriction digests are collected on agarose gels. Then, using a strategy similar to that used by Coulson et al (1986), we measure the relative mobilities of restriction fragments and use these to identify other clones that share a large proportion of fragments with the same relative mobilities, plus or minus a constant ''tolerance''.…”

“…Furthermore, no information on clone size is recovered, and the absence of predictable signal intensity from band to band presents significant challenges for fully automated band calling. Another method under development is the multiple-complete-digest (MCD) mapping (Wong et al 1997) in which three separate restriction digestions of a cosmid clone are analyzed by agarose gel electrophoresis and the data are used to construct a detailed restriction map. Here, the developers have not relied on the universally available BAC and PAC libraries, instead constructing custom cosmid libraries from redundant YACs.…”