Low cost, highly efficient library preparation
Echo Liquid Handlers enable library preparation in low microliter volumes for a range of sequencing methods including Sanger and next-generation sequencing (NGS) – dramatically reduce reagent costs, save samples, and eliminate steps—all while improving library quality.
When NGS sequencing programs scale, the cost of sample preparation is a limiting factor. The Echo Liquid Handlers offer an opportunity to scale with confidence. By combining the Echo Liquid Handler's low-volume acoustic transfer capability with Illumina's Nextera XT library preparation kit, researchers can drastically increase the number of libraries generated. Whether using the Nextera XT kit for amplicon, plasmid, or small genome sequencing, the Echo system can reduce transfer volumes by 75% or more. The same benefits can be realized with sample preparation kits from NuGEN, NEB, and others.
Echo Liquid Handlers can rapidly pool and normalize oligos or DNA libraries from any well of a microplate. With average transfer times of no more than a few seconds per well. Echo Liquid Handlers can reduce pooling times by more than 80% compared to traditional liquid handlers. Since Echo systems can transfer volumes as little as 2.5 nanoliters, high concentration libraries do not have to be diluted prior to transfer. This capability results in simultaneous normalization while pooling. Furthermore, by avoiding tip-based sample dilution, the propagation of errors from sample retention by tips is eliminated. This drastically improves the concentration accuracy of all libraries pooled.
by Amyris, Inc.
Elaine B. Shapland, Victor Holmes, Christopher D. Reeves, Elena Sorokin, Maxime Durot*, Darren Platt, Christopher Allen, Jed Dean, Zach Serber, Jack Newman, and Sunil Chandran
Amyris Inc., *TOTAL New Energies USA, Inc.
ABSTRACT: In recent years, next-generation sequencing (NGS) technology has greatly reduced the cost of sequencing whole genomes, whereas the cost of sequence verification of plasmids via Sanger sequencing has remained high. Consequently, industrialscale strain engineers either limit the number of designs or take short cuts in quality control. Here, we show that over 4000 plasmids can be completely sequenced in one Illumina MiSeq run for less than $3 each (15× coverage), which is a 20-fold reduction over using Sanger sequencing (2× coverage). We reduced the volume of the Nextera tagmentation reaction by 100-fold and developed an automated workflow to prepare thousands of samples for sequencing. We also developed software to track the samples and associated sequence data and to rapidly identify correctly assembled constructs having the fewest defects. As DNA synthesis and assembly become a centralized commodity, this NGS quality control (QC) process will be essential to groups operating high-throughput pipelines for DNA construction.
|Manual Pipetting||Echo® Liquid Handler|
|Amount of DNA||50 ng||0.06 – 2.0 ng|
|DNA volume (Rxn)||25 µL||200 nL|
|Library prep volume (Rxn)||25 µL||300 nL|
|Total volume||50 µL||0.5 µL|
|Reactions per kit||96||9600|
|Cost per reaction||$72.91||$0.73|
FEATURED APPLICATION NOTE
The ongoing evolution of next-generation sequencing (NGS) technologies has reduced sequencing costs by orders of magnitude and signi cantly increased throughput. As the cost of NGS has decreased, library preparation costs have become a more signi cant proportion of the total cost. Miniaturizing sample preparation volumes provides the opportunity for dramatic reagent cost-savings. Here, we investigate two possible approaches to increase cost-saving in library preparation work ows which are enabled by the use of the Labcyte Echo 525 Acoustic Liquid Handler.