Pooled genomic indexing (PGI). BACs are arrayed in a conceptual rectangular array (top). The original glycerol stock is used to inoculate the Original Master array, which is then grown and used to inoculate two sets of plates, one used to grow cultures for pooling by row and another for pooling cultures by column. For sufficient yields of cells, BACs were grown in duplicate plates, as described in the Methods section. Striped arrows indicate inoculation and growth of cultures in the 96-well format. A shuffled array is also inoculated by using a specially designed single-tip rearraying robot. Shotgun libraries are constructed for each row- and column-pool. Reads from the pools are deconvoluted to the original BACs using the reference genomic sequence (bottom left). If reads from at least two pools map close to each other within the reference sequence, the reads are deconvoluted to the BAC at the intersection. The BAC is also mapped onto the segment (index) between the mapped locations. ST-PGI (bottom right) sequences multiple, short, mapable sequence tags of length between 50 and 200 bp within a single reaction.