Hybsweeper: a resource for detecting high-density plate gridding coordinates

Cosmid (1), phage, and bacterial artificial chromosome (BAC) libraries (2) have commonly been used for constructing genome libraries, and depending on the organism, this results in thousands of clones that need to be managed. One complication of dealing with such a high number of samples arises during hybridization screening of high-density filter arrays of the genomic clones and trying to distinguish the correct dishes and wells of positive signals. There exists a need for easy determination of clone identities from autoradiographs generated through simple probing methods, overgo probe hybridization (3), and isolation of unique clones from saturation hybridization. To meet this need, we have developed Hybsweeper, a program that is designed to allow quick mapping of positive spots on a filter and straightforward identification of the corresponding clone. We demonstrate the utility of Hybsweeper by application to an established wheat genome library. The five-times coverage genome library of the 13,500 Mb tetraploid genome of Triticum turgidum subsp. durum is represented by 516,256 BAC clones averaging approximately 130 kb per insert (2). Management of clones for plate replication and spot arraying was provided by the QBot (Genetix, Hampshire, UK), a multifunctional replication robot system. The tested clone library collection was distributed over 1344 384-well microtiter dishes (516,096 clones). As only 48 384-well microtiter dishes could be plated on the QBot apparatus at a time, this collection was enough to create 28 reading sets of the 67,092 Mb of insert DNA, assuming an average of 130 kb/ insert. To facilitate clone retrieval, clones were replicated onto nylon filters where cells were incubated for 14 h, lysed, and prepared for DNA-DNA hybridization experiments (2). The gridding pattern used was one provided by the software included with the QBot system and consisted of 48 384-well plates replicated over six gridding areas (eight 384-well plates per gridding area). Each gridding area consists of a 16 × 24 array of a 4 × 4 stamped pattern of clones. Each well of a represented plate was double stamped within the 16 spots using an offset 4 × 4 stamping pattern. Eight different doublet-patterns were used to allow identification of of the correct plate from among the eight possible. This double-stamping within a 4 × 4 arrayed area was important in reducing errors in spot picking. To identify genomic clones that contained genes of interest, several representative gene clones were radioactively labeled and hybridized to the genomic clones …