title: Accuracy and quality assessment of 454 GS-FLX Titanium pyrosequencing.
abstract: BACKGROUND
  The rapid evolution of 454 GS-FLX sequencing technology has not been accompanied by a reassessment of the quality and accuracy of the sequences obtained. Current strategies for decision-making and error-correction are based on an initial analysis by Huse et al. in 2007, for the older GS20 system based on experimental sequences. We analyze here the quality of 454 sequencing data and identify factors playing a role in sequencing error, through the use of an extensive dataset for Roche control DNA fragments.
  
  
  RESULTS
  We obtained a mean error rate for 454 sequences of 1.07%. More importantly, the error rate is not randomly distributed; it occasionally rose to more than 50% in certain positions, and its distribution was linked to several experimental variables. The main factors related to error are the presence of homopolymers, position in the sequence, size of the sequence and spatial localization in PT plates for insertion and deletion errors. These factors can be described by considering seven variables. No single variable can account for the error rate distribution, but most of the variation is explained by the combination of all seven variables.
  
  
  CONCLUSIONS
  The pattern identified here calls for the use of internal controls and error-correcting base callers, to correct for errors, when available (e.g. when sequencing amplicons). For shotgun libraries, the use of both sequencing primers and deep coverage, combined with the use of random sequencing primer sites should partly compensate for even high error rates, although it may prove more difficult than previous thought to distinguish between low-frequency alleles and errors.
authors: Aix-Marseille Université, CNRS, IRD, UMR 6116 - IMEP, Equipe Evolution Génome Environnement, Centre Saint-Charles, Case 36, 3 place Victor Hugo, 13331 Marseille Cedex 3, France.
authors: André
authors: A
authors: Gilles
authors: Emese
authors: E
authors: Meglécz
authors: Nicolas
authors: N
authors: Pech
authors: Stéphanie
authors: S
authors: Ferreira
authors: Thibaut
authors: T
authors: Malausa
authors: Jean-François
authors: JF
authors: Martin
references: Genetica. 2010 Apr;138(4):433-51
references: 18931921
references: Bioinformatics. 2007 Nov 1;23(21):2947-8
references: 17846036
references: Environ Microbiol. 2010 Jan;12(1):118-23
references: 19725865
references: Nat Rev Genet. 2010 Jan;11(1):31-46
references: 19997069
references: Environ Microbiol. 2010 Jul;12(7):1889-98
references: 20236171
references: Proc Natl Acad Sci U S A. 2008 Sep 2;105(35):13081-6
references: 18723673
references: Genomics. 2007 Sep;90(3):291-6
references: 17587543
references: Genetics. 2009 May;182(1):295-301
references: 19293142
references: BMC Genomics. 2010 May 11;11:296
references: 20459828
references: Genome Biol. 2007;8(7):R143
references: 17659080
references: Nature. 2005 Sep 15;437(7057):376-80
references: 16056220
references: Nat Methods. 2008 Feb;5(2):179-81
references: 18193056
references: PLoS Comput Biol. 2009 Sep;5(9):e1000502
references: 19750212
references: PLoS One. 2010 Apr 06;5(4):e10029
references: 20386591
references: Mol Ecol. 2009 May;18(9):1818-20
references: 19317846
references: Mol Biol Evol. 2008 Nov;25(11):2409-19
references: 18725384
references: Nucleic Acids Res. 2008 Sep;36(16):e105
references: 18660515
references: BMC Genomics. 2008 Dec 16;9:603
references: 19087275
references: BMC Genomics. 2009 May 19;10:234
references: 19454017
references: Sci China Life Sci. 2010 Jan;53(1):44-57
references: 20596955
references: Nucleic Acids Res. 1997 Sep 1;25(17):3389-402
references: 9254694
references: BMC Genomics. 2009 Nov 12;10:520
references: 19909532
references: Mol Ecol Resour. 2009 May;9(3):713-9
references: 21564729
references: Breast Cancer Res. 2009;11 Suppl 3:S12
references: 20030863