title: Increased throughput by parallelization of library preparation for massive sequencing.
abstract: 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.
  
  
  RESULTS
  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
  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.
authors.affiliation: Division of Gene Technology, School of Biotechnology, Royal Institute of Technology (KTH), Stockholm, Sweden.
authors.forename: Sverker
authors.initials: S
authors.lastname: Lundin
authors.forename: Henrik
authors.initials: H
authors.lastname: Stranneheim
authors.forename: Erik
authors.initials: E
authors.lastname: Pettersson
authors.forename: Daniel
authors.initials: D
authors.lastname: Klevebring
authors.forename: Joakim
authors.initials: J
authors.lastname: Lundeberg
mesh_terms: D001331:Automation; D002264:Carboxylic Acids; D011232:Chemical Precipitation; D015723:Gene Library; D011092:Polyethylene Glycols; D017422:Sequence Analysis, DNA / Q000295:instrumentation / Q000379:methods*
chemical_list: D002264:Carboxylic Acids; D011092:Polyethylene Glycols
references.citation: Nat Biotechnol. 2009 Nov;27(11):1013-23
references.pmid: 19898456
references.citation: Nucleic Acids Res. 1995 Nov 25;23(22):4742-3
references.pmid: 8524672
references.citation: Nucleic Acids Res. 2009 May;37(8):e63
references.pmid: 19304748
references.citation: Methods Enzymol. 1980;65(1):347-53
references.pmid: 6246357
references.citation: Nature. 2005 Sep 15;437(7057):376-80
references.pmid: 16056220
references.citation: Anal Biochem. 2008 Sep 15;380(2):310-4
references.pmid: 18571491
references.citation: Anal Biochem. 1993 Jan;208(1):171-5
references.pmid: 8382019
references.citation: Nucleic Acids Res. 2008 Jan;36(1):e5
references.pmid: 18084031
references.citation: Nucleic Acids Res. 1991 Mar 25;19(6):1346
references.pmid: 2030954
references.citation: Anal Biochem. 2006 Jul 15;354(2):311-3
references.pmid: 16725102
references.citation: Nat Methods. 2008 Dec;5(12):1005-10
references.pmid: 19034268
references.citation: Curr Protoc Hum Genet. 2009 Apr;Chapter 18:Unit18.1
references.pmid: 19360698
references.citation: Nucleic Acids Res. 1975 Mar;2(3):383-9
references.pmid: 236548
references.citation: Genomics. 2009 Feb;93(2):105-11
references.pmid: 18992322
references.citation: Nat Protoc. 2008;3(2):267-78
references.pmid: 18274529
references.citation: Biotechniques. 2009 Jan;46(1):51-2, 54-7
references.pmid: 19301622
references.citation: J Biomed Biotechnol. 2009;2009:396808
references.pmid: 19841682
references.citation: Anal Biochem. 1996 Nov 1;242(1):84-9
references.pmid: 8923969