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Applications that use Bacterial Artificial Chromosome (BAC) libraries often require paired-end sequences and knowledge of the physical location of each clone in plates. To facilitate obtaining this information in high-throughput, we generated pBACode vectors: a pool of BAC cloning vectors, each with a pair of random barcodes flanking its cloning site. In a pBACode BAC library, the BAC ends and their linked barcodes can be sequenced in bulk. Barcode pairs are determined by sequencing the empty pBACode vectors, which allows BAC ends to be paired according to their barcodes. For physical clone mapping, the barcodes are used as unique markers for their linked genomic sequence. After multi-dimensional pooling of BAC clones, the barcodes are sequenced and deconvoluted to locate each clone. We generated a pBACode library of 94,464 clones for the flounder Paralichthys olivaceus and obtained paired-end sequence from 95.4% of the clones. Incorporating BAC paired-ends into the genome preassembly improved its continuity by over 10-fold. Furthermore, we were able to use the barcodes to map the physical locations of each clone in just 50 pools, with up to 11 808 clones per pool. Our physical clone mapping located 90.2% of BAC clones, enabling targeted characterization of chromosomal rearrangements.

The public project's sequencing strategy involved producing a map of the human genome, and then pinning sequence to it. This helps to avoid errors in the sequence, especially in repetitive regions.

The sequence of nucleotides within a DNA molecule can yield a wealth of information. Important biological features such as amino acid sequence, exact amino acid composition, and gene structure are contained within sequences. The data can be used for highly specific manipulations of cloned DNA, such as changing a single nucleotide, substituting promoters between genes, and fusing of genes to yield hybrid (heterologous) proteins. Probes can also be designed for a variety of uses, including the detection of genetic diseases and pathogenic microbes.

Products for recombinant DNA studies described this week include a biotin clone detection kit and a system for dissecting yeast asci.