Roche 454 Service Details
Project and Assay Design Workflow
Initially, a meeting will be planned to discuss your research goals and the suitability of the Roche 454 technology in achieving those goals. If this platform is applicable, you will submit and get approval a CSAS request; once a CSAS is approved, reagents will be ordered from Roche and we will begin processing your samples.
Sample Processing Workflow
Once samples are received, LMT staff determines the concentration via either NanoDrop and Flash Gel (Shotgun) or Bioanalyzer DNA1000 chip (Amplicon). You will be notified if your samples do not pass this initial QC step. If the project involves targeted regions of samples in multiples of 48 (samples and regions), amplicons can be generated on Fluidigm’s Access Array. Amplicon libraries are then prepared using Ampure, a high sensitivity QC chip, a qPCR quantitation, and equimolar pooling. Shotgun rapid library preparations include fragmentation, end repair, adapter ligation, Ampure, and a high sensitivity QC chip. Medium or large volume emulsion pcr is performed using either Lib-L or Lib-A. Breaking and enriching is performed, followed by a FLX Titanium sequence run on the PTP (Pico Titer Plate). Sequence run data metrics are reviewed on the Wiki LIMS and sent to the Advanced Biomedical Computing Center (ABCC) for further QC. The customer is notified from ABCC when they have data to view on the 454 Wiki LIMS.