Automation of the GS FLX Shotgun Library Preparation
DNA library preparation is the entry point of the 454 Sequencing™ sample preparation process for many applications. The current protocol is amenable for parallel processing of a large number of samples by one individual. We have adapted the GS FLX shotgun DNA library protocol to allow the parallel processing of up to 96 libraries using a commercially available liquid handling automation.
Nebulization has been replaced by fragmentation on a Covaris™ E210 instrument. This allows for the unattended fragmentation of up to 96 samples in 6.5 hours. Because there is no DNA loss during fragmentation with the Covaris™ instrument we have been able to reduce the DNA input requirement from 5 to 3 ug of double stranded DNA. All post-fragmentation steps are carried out in a 96-well plate format on a Hamilton MICROLAB® STAR liquid handler. All Qiagen® MinElute® clean ups have been eliminated and replaced by purification using a combination of Agencourt AMPure® SPRI® beads and Qiagen® QIAquick® 96 well plate. The processing time from sizing SPRI® to single stranded library takes approximately 5 hours.
The automated processing appears robust and reproducible. We have also validated the method with Multiplexing Identifiers (MIDs). We find library quality to be equivalent to the manual method. The method is currently in production at the 454 Sequencing Center.
- We have successfully automated the 454 Sequencing™ FLX library preparation protocol.
- We have replaced labor intensive manual nebulization by fragmentation on a Covaris™ E210 instrument.
- We have adapted the FLX library protocol to make it compatible with standard liquid handling automation.
- OTS components used: No custom equipment or instrumentation.
- Processing of up to 96 samples can be done in parallel over a 2-day period
- Fragmentation of 96 samples takes 6.5 hours
- Processing samples through the rest of library prep takes 5 hours
- STAR 8/96 AL iSWAP
- Multiflex CO-RE Gripper
- Vector 4.1
- STAR Cabinet
- Tip Carrier, 5 Tip Racks
- Deepwell Carrier
- L4 Carrier
- Inheco CPAC
- Inheco, LPAC, UF Plate Heater
- Adapter Plate for CPAC
- Control Cable
- Driver, Inheco Hotel/Variomag
- BVS, 4 Liter Waste Assembly
- STAR Tip 50UL Cond
- CORE Tips 300UL
- Core Tips 1 ML
- CPAC Trough Insert + 7T Mod
- TEC Controller