From Macro to Micro—Automated Microfluidic Sanger Sequencing
Microfluidic Sanger sequencing is a lab-on-a-chip application for DNA sequencing. Samples are typically in milliliter volumes—the ‘macro world’—and must be interfaced to a microfluidic system that handles only microliters. The process is relatively expensive when performed manually due to the expense of the Big Dye and the reagents required for the clean-up method. However, by integrating microfluidic technology with a highly reliable robotic system, reagent volumes can be drastically reduced, generating substantial cost savings.
For laboratories performing Sanger sequencing, a major ‘pain point’ has always been the expense of the Big Dye, XTerminator, and magnetic bead reagents required for cleanup. While significantly cheaper, ethanol clean-up is a manual technique that is both tedious and time consuming. With a clear understanding of microfluidic technology and the sequencing market, it became obvious that this knowledge could be used to develop an automated sequencing system that would drastically reduce the reagent volumes required for efficient clean-up. The initial outlay for such a system is more than offset by the generation of substantial cost savings through reduced reagent consumption.
Two issues needed to be resolved to produce an automated sequencing platform. Typically samples are presented in large volumes—milliliters—that are easy to handle and manipulate. These large volume samples, known as the ‘macro world,’ must be interfaced to a microfluidic system that handles only microliter volumes – the ‘micro world’. The transfer of samples from macro to micro also required the integration of a highly reliable and robust robotic platform into the IntegenX Apollo 100XL system, and the Company chose Tecan’s Cavro Mini Sample Processor (MSP) 9250 robot, equipped with an 8-channel probe assembly and washable stainless steel tips.
The work deck of the robotic platform was extended to accommodate sample microplates, product plates and reagents, and the microfluidic station, where up to four MOVe microfluidic chips can be mounted. The microfluidic system features built-in thermocyclers and a series of microfluidic valves configured to act as pumps, controlling the direction of flow and pumping reagents around the microchip for magnetic bead clean-up. Processed samples are then transferred into the product plate, ready for sequencing.
The dedicated sample preparation set-up offers a number of advantages and cost savings for the laboratory. Reducing the reaction volumes to approximately 1µl significantly decreases the amount of precious sample that is required for the sequencing process. Accordingly, the amount of Big Dye and magnetic beads are reduced, substantially lowering the cost per sample. The system has the capacity to run 96 samples at a time, and provides full walkaway automation, which frees valuable staff time for other tasks, eliminating potential human errors that could lead to expensive repeat runs.
Typically, researchers submitting samples before noon will expect to receive their results by noon the following day. With the complete cycle taking four hours, including a 30 minute chip cleaning procedure at the end of the cycle, the user is able to process two plates a day. Throughput can be further increased, compared to a manual method, by running a third plate overnight. The automated sequencing system uses an Active Temperature Control unit, which keeps samples completely stable and ready to be placed in the sequencer in the morning. The Active Temperature Control unit is a big advantage when urgent samples are received too late in the day to be processed manually, as researchers will still be able to receive the data before noon the next day.
Automated sequencing platforms offer clear benefits to the laboratory, and additional applications for this technology—potentially next generation sequencing—are being considered for the future.
James Nelson is Marketing Director at IntegenX, Inc. He can be reached at 925-701-3400 or email@example.com.