A new implementation of high-throughput five-dimensional clone pooling strategy for BAC library screening

Abstract

Background: A five-dimensional (5-D) clone pooling strategy for screening of bacterial artificial chromosome (BAC) clones with molecular markers utilizing highly-parallel Illumina GoldenGate assays and PCR facilitates high-throughput BAC clone and BAC contig anchoring on a genetic map. However, this strategy occasionally needs manual PCR to deconvolute pools and identify truly positive clones.

Results: A new implementation is reported here for our previously reported clone pooling strategy. Row and column pools of BAC clones are divided into sub-pools with 1~ 2 x genome coverage. All BAC pools are screened with Illumina's GoldenGate assay and the BAC pools are deconvoluted to identify individual positive clones. Putative positive BAC clones are then further analyzed to find positive clones on the basis of them being neighbours in a contig. An exhaustive search or brute force algorithm was designed for this deconvolution and integrated into a newly developed software tool, FPCBrowser, for analyzing clone pooling data. This algorithm was used with empirical data for 55 Illumina GoldenGate SNP assays detecting SNP markers mapped on Aegilops tauschii chromosome 2D and Ae. tauschii contig maps. Clones in single contigs were successfully assigned to 48 (87%) specific SNP markers on the map with 91% precision.

Conclusion: A new implementation of 5-D BAC clone pooling strategy employing both GoldenGate assay screening and assembled BAC contigs is shown here to be a high-throughput, low cost, rapid, and feasible approach to screening BAC libraries and anchoring BAC clones and contigs on genetic maps. The software FPCBrowser with the integrated clone deconvolution algorithm has been developed and is downloadable at http://avena.pw.usda.gov/wheatD/fpcbrowser.shtml.

Figure 1

Schematic display of the deconvolution of 5-D clone pools with the Illumina GoldenGate assay and a contig map. A total of 302,976 BAC clones of Ae. tauschii were screened for the SNP marker BE442608. In the super pool screening step, 5 row hits and 6 column hits were observed and thus 30 potential plate hits were obtained. To screen plate rows and plate columns, the entire stack of plates was split into 5 smaller stacks (sub-pools) with only 1~2× coverage based on individual BAC libraries (see Table 2) and those sub-pools (RP_HB and CP_HB, RP_HI and CP_HI, RP_HD and CP_HD, and RP_BB and CP_BB) were screened separately, dramatically decreasing the number of candidate positive clones and F+ clones. By combining observed plate hits and row/column hits for each sub-pools, a total of 546 candidate clones (144, 24, 42 and 336 candidate clones for HB, HI, HD and BB sub-pools, respectively) were obtained. Of the candidate clones, 362 were located in 336 contigs, 336 clones were not included in contig assembly because of substandard or failed fingerprints, and the remaining 23 clones were singletons. The deconvolution algorithm was used to detect truly positive (TP) clones among the 362 candidate clones. Five TP clones, BB092N18, BB070C1, HB086J7, HI137N13, and HB006B8 were detected in ctg4985. The SNP marker derived from EST BE442608 was previously mapped on the genetic map of Ae. tauschii chromosome 2D, hence contig ctg4985 was anchored at this locus on the 2D genetic map. This inference agrees with the previous anchoring of contig ctg4985 [9].

Figure 2

False positive (F+) pool hit rate and false negative (F-) pool hit rate with clone deconvolution. (A) Clone row pool (RP) and column pool (CP) F+ rate; (B) RP and CP F- rate; (C) Row super pool (RSP) and column super pool (CSP) F+ rate; and (D) RSP and CSP F- rate. The F+ rate of a pool for a marker is calculated as F+ hits divided by observed pool hits, and the F- rate of a pool for a marker as F- pool hits divided by true pool hits. Based on a given list of true positive BAC clones associated with a marker, true pool hits of CP, RP, CSP and RSP can be reversely obtained from information extracted from clone names. If a true pool hit is not found in the observed pool hits, the hit is F-. However, if an observed pool hit does not exist in the true pool hits, the hit is F+.

Figure 3

Screen shot of the FPCBrowser software with the clone deconvolution module running.

Figure 4

True positive (TP) clones associated with a molecular marker in a contig map. Several scenarios were encountered when clones were screened by PCR[9]. (A) All TP clones associated with a marker shared a DNA fragment and had therefore a spanning or inclusion relations. (B) No common DNA fragment was observed in all TP clones associated with a marker but two or more contiguous clones had fingerprint overlaps. (C), (D), and (E) All TP clones associated with a marker were grouped to more than one contig (D), or a contig and singleton (C), or all TP clones were singletons (E). In scenarios C, D, and E, some of the TP clones were usually located at the end of one contig and the rest were located at the end of another contig, or were singletons. The cause of these scenarios was the high stringency used in contig assembly that resulted in two separated contigs or in a contig and a singleton.