Figure 1. Cartesian Pooling-Coordinate Sequencing (CP-CSeq) concept for simultaneously determining both tag sequence and library coordinates of the biological entity containing the tag sequence.

Here, the optimized concept is presented in the case of M. bovis BCG transposon-tagged mutants. (a) Layout and Cartesian Pooling of a 96 × 96-well library of sequence-tagged biological entities (for example, M. bovis BCG transposon insertion mutants). Each entity's position in the library is characterized by three Cartesian coordinates (X, Y and Z). To create the X–Y Pool Plate, a small culture volume of one specific well position (for example, A1) in all of the 96-well plates was transferred to and thus pooled in the same specific well (for example, A1) of the masterplate, thus keeping the respective positions within each primary plate (X and Y coordinates). Subsequently, the Z pool plate was prepared by pooling all of the 96 wells of a primary plate in one single well of the masterplate (Z-coordinate). Next, each row and each column of both masterplates were pooled in column (n=12) and row (n=8) pools, giving a total of 40 samples that represent the 96 × 96 clone library. (b) Coordinate-Seq sequencing library preparation links a pool-specific barcode to the sequence tag that identifies each biological entity in each pool. Here, the protocol is optimized for Himar1 transposon-flanking sequence tags. (c) Coordinate-Seq sequence data processing for transposon insertion mutants.