Increased Throughput by Parallelization of Library Preparation for Massive Sequencing

Background Massively parallel sequencing systems continue to improve on data output, while leaving labor-intensive library preparations a potential bottleneck. Efforts are currently under way to relieve the crucial and time-consuming work to prepare DNA for high-throughput sequencing. Methodology/Principal Findings In this study, we demonstrate an automated parallel library preparation protocol using generic carboxylic acid-coated superparamagnetic beads and polyethylene glycol precipitation as a reproducible and flexible method for DNA fragment length separation. With this approach the library preparation for DNA sequencing can easily be adjusted to a desired fragment length. The automated protocol, here demonstrated using the GS FLX Titanium instrument, was compared to the standard manual library preparation, showing higher yield, throughput and great reproducibility. In addition, 12 libraries were prepared and uniquely tagged in parallel, and the distribution of sequence reads between these indexed samples could be improved using quantitative PCR-assisted pooling. Conclusions/Significance We present a novel automated procedure that makes it possible to prepare 36 indexed libraries per person and day, which can be increased to up to 96 libraries processed simultaneously. The yield, speed and robust performance of the protocol constitute a substantial improvement to present manual methods, without the need of extensive equipment investments. The described procedure enables a considerable efficiency increase for small to midsize sequencing centers.

[1]  James R. Knight,et al.  Genome sequencing in microfabricated high-density picolitre reactors , 2005, Nature.

[2]  D. G. Wang,et al.  Solid-phase reversible immobilization for the isolation of PCR products. , 1995, Nucleic acids research.

[3]  J. Lundeberg,et al.  Automation of cDNA Synthesis and Labelling Improves Reproducibility , 2009, Journal of biomedicine & biotechnology.

[4]  Richard Durbin,et al.  A large genome center's improvements to the Illumina sequencing system , 2008, Nature Methods.

[5]  D. Riesner,et al.  Purification of nucleic acids by selective precipitation with polyethylene glycol 6000. , 2006, Analytical biochemistry.

[6]  Matthias Meyer,et al.  From micrograms to picograms: quantitative PCR reduces the material demands of high-throughput sequencing , 2007, Nucleic acids research.

[7]  M. Uhlén,et al.  Solid phase DNA minisequencing by an enzymatic luminometric inorganic pyrophosphate detection assay. , 1993, Analytical biochemistry.

[8]  M. Ronaghi,et al.  Real-time DNA sequencing using detection of pyrophosphate release. , 1996, Analytical biochemistry.

[9]  Joakim Lundeberg,et al.  Generations of sequencing technologies. , 2009, Genomics.

[10]  Patrik L. Ståhl,et al.  Flow cytometry for enrichment and titration in massively parallel DNA sequencing , 2009, Nucleic acids research.

[11]  J. Lis.  Fractionation of DNA fragments by polyethylene glycol induced precipitation. , 1980, Methods in enzymology.

[12]  K. Paithankar,et al.  Precipitation of DNA by polyethylene glycol and ethanol. , 1991, Nucleic acids research.

[13]  B. Roe,et al.  Methods for Generating Shotgun and Mixed Shotgun/Paired‐End Libraries for the 454 DNA Sequencer , 2009, Current protocols in human genetics.

[14]  R. Schleif,et al.  Size fractionation of double-stranded DNA by precipitation with polyethylene glycol. , 1975, Nucleic acids research.

[15]  Timothy D. Harris,et al.  The challenges of sequencing by synthesis , 2009, Nature Biotechnology.

[16]  S. Pääbo,et al.  Optimization of 454 sequencing library preparation from small amounts of DNA permits sequence determination of both DNA strands. , 2009, BioTechniques.

[17]  U. Stenzel,et al.  Parallel tagged sequencing on the 454 platform , 2008, Nature Protocols.

[18]  Marie-Laure A. Sauer,et al.  Sequential CaCl2, polyethylene glycol precipitation for RNase-free plasmid DNA isolation. , 2008, Analytical biochemistry.