Lawrence Berkeley National Laboratory

As the demand of constructing Illumina libraries increases, we have started to modify the library construction protocol to adapt the use of multichannel pipette and 96-well plates. With the few simple modification steps, we have doubled the library production efficiency. These modifications include the shearing of DNA with Covaris E210, and the cleaning of enzymatic reactions and fragment size selection with SPRI beads and a magnetic plate holder. We have also designed a set of molecular barcodes to enable the sequencing of many libraries in parallel. The requirements of these barcodes include 4 bases, balanced GC, and at least 2 bases difference between barcodes. The barcode is attached to the adaptor so it does not require third sequencing primer and the barcoded library can be run on the same flowcell/run with other non-barcoded libraries. We have begun to assess the ability to assign reads and the potential bias towards certain barcodes after pooling different number of libraries. We have recently programmed the Biomek FX robot to carry out the library construction process. Although this process still require manual transfer of plates from robot to other work stations, the processing of 96 Illumina libraries takes approximately 6-8 hours. This semi-automated process represents a significant increase of library capacity comparing to the manual process. We will present the progress and the challenges of these scale-up processes.