Mission: Gaps no more

Broad Institute researchers employ Roche-454 technology to close some gaps in the human genome that resist traditional bacterial cloning methods

Jeffrey Bouley
CAMBRIDGE, Mass.—While the Human Genome Project has done a great deal to advance genomics, and many up-and-coming technologies present the possibility of relatively affordable whole-genome sequencing in the near future, things still aren't perfect, as evidenced by the fact that the most recent release of the finished human genome contains 260 euchromatic gaps, excluding chromosome Y. Researchers at the Broad Institute of MIT and Harvard may have found a simple and easily scalable method to close nearly half of these gaps, using the GS-20 FLX instrument from Roche's 454 Life Sciences division.

In their article, "Closing gaps in the human genome using sequencing by synthesis," published in BioMed Central's open-access journal Genome Biology, the Broad team noted that there are three classes of gaps. Type I gaps are subtelomeric, with nine gaps in subtelomeric regions containing telomere-associated repeats, while type II gaps contain duplicated euchromatin—this includes 30 pericentromeric gaps and 94 gaps flanked by segmental duplications. Both of those types of gaps are "structural" gaps according to the Broad research team, which notes that they "arise from unresolved structural complexity in the genome and can be attacked by the methodology of carefully reassembling existing tiling paths or by reassembling the area using a single haplotype."

The remaining gaps, however, are in unique euchromatin, and these 127 "non-structural" gaps remain problematic because they contain sequences that are unstable—perhaps even toxic—to the standard bacterial cloning methods used for libraries in the Human Genome Project, leaving a high bias for them to be deleted by bacterial clones.

It was for those 127 type III gaps that the Broad research team applied the sequencing technology of 454 Life Sciences, closing all three of the remaining non-structural gaps in chromosome 15 as their proof of principle.

Clone-based methods "remain an effective means of attacking structural gaps," notes Manuel Garber, a computational biologist at the Broad Institute, and one of the lead members of the research team. However, bacterial cloning likely won't resolve the 127 gaps in the non-structural type III class.

That was the reason the team choose to use the 454 methodology, because unlike traditional sequencing, it has no bacterial cloning step. Garber and colleagues note that previous reports has suggested that some of the non-structural gaps might also be closed using "extreme" methods, such as cloning in yeast, but they point out that such methods are laborious and less scalable than the 454 approach.

"The technique we present could be also be applied to the targeted closure of gaps in other finished or near finished genomes such as mouse and dog, which contain 103 and 47 class III gaps, respectively," Garber notes.

"In the end," he says, "I think what our work really tells us is that a mix of technologies and approaches is what will give the best results and the best genomes possible."

Jeffrey Bouley

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