We perform about 1500 BRCA1/2 samples/year by MPS. In this context, library preparation for MPS is one of the most critical, on bench and time-consuming steps our pipeline. In addition, automation of library preparation phase is the best option to avoid the risk of human-introduced error. Our objective was to report a rapid, automated solution to prepare Multiplicom-NGS based protocol for Illumina MiSeq® sequencer for BRCA1/2 genes and to analyze the performance of MPS in terms of number of sequences/run, coverage uniformity and number of variants detected. A total of 120 samples were used to evaluate the automated preparative process.
Complete protocol for BRCA1/2 genes amplification by the BRCA MASTR Dx kit (Multiplicom, Niel, Belgium) was performed using OMNIA LH 100 and OMNIA LH 75 automated workstations designed and produced by MASMEC Biomed (Modugno, Bari). The LH 100 was equipped with a robot (X-Y-Z), 8 independent pipette channels and a layout with two racks for reagents and DNA samples, 9 deck positions for 96 well plates and different size tips and two heating-cooling units for controlled temperature steps. The LH 75 was prepared with a single pipette and a magnetic tool for analyzing 12 samples at the same time and 6 deck positions for 96 well plate and different size tips. Both workstations were controlled by MASMEC Framework software and were provided with UV lamp for decontamination to reduce the risk of crosscontamination.
OMNIA platforms were accurately customized to set up libraries preparation and purification process of 12 patients simultaneously per run in only 8-9 working hours (respect to 1-2 working days for manual execution). Because the workstations require very little hands-on time, 12 consecutive samples were prepared for the next automated amplification cycle while the first 12 samples were purified. Thanks to perfect setting of ratio between magnetic beads and DNA amount, a high-quality of BRCA1/2 libraries and MPS performance were obtained.
This study describes a new automated solution for fast and reproducible BRCA1/2 library preparation for MPS using a robotic workstation. Automated library preparation and MPS performance were comparable to a standard manual library preparation. The throughput of our pipeline was high positively improved by introducing these machines in our routine workflow.
Authors: M. De Bonis, A. Costella, L. Gentile, A. Minucci, E.D. Capoluongo
Laboratory of Clinical Molecular and Personalized Diagnostics, Institute of Biochemistry and Clinical Biochemistry, Catholic University, Rome, Italy
for more information on the BRCA MASTR Dx.