Plant & Animal Genomes XVII Conference

January 10-14, 2009
Town & Country Convention Center
San Diego, CA

Automation Of The GS FLX Shotgun Library Preparation And Implementation At The 454 Sequencing Center

Darren H. Birr , Ethan DeFord , Cynthia L. Turcotte , Pascal Bouffard , Gerald P. Irzyk

  454 Life Sciences Corporation, A Roche Company, 1 Commercial Street, Branford, CT 06405

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 Nebulization has been replaced by fragmentation on a Covaris™ E210 instrument.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.