title: Assessment of whole genome amplification-induced bias through high-throughput, massively parallel whole genome sequencing.
abstract: BACKGROUND
  Whole genome amplification is an increasingly common technique through which minute amounts of DNA can be multiplied to generate quantities suitable for genetic testing and analysis. Questions of amplification-induced error and template bias generated by these methods have previously been addressed through either small scale (SNPs) or large scale (CGH array, FISH) methodologies. Here we utilized whole genome sequencing to assess amplification-induced bias in both coding and non-coding regions of two bacterial genomes. Halobacterium species NRC-1 DNA and Campylobacter jejuni were amplified by several common, commercially available protocols: multiple displacement amplification, primer extension pre-amplification and degenerate oligonucleotide primed PCR. The amplification-induced bias of each method was assessed by sequencing both genomes in their entirety using the 454 Sequencing System technology and comparing the results with those obtained from unamplified controls.
  
  
  RESULTS
  All amplification methodologies induced statistically significant bias relative to the unamplified control. For the Halobacterium species NRC-1 genome, assessed at 100 base resolution, the D-statistics from GenomiPhi-amplified material were 119 times greater than those from unamplified material, 164.0 times greater for Repli-G, 165.0 times greater for PEP-PCR and 252.0 times greater than the unamplified controls for DOP-PCR. For Campylobacter jejuni, also analyzed at 100 base resolution, the D-statistics from GenomiPhi-amplified material were 15 times greater than those from unamplified material, 19.8 times greater for Repli-G, 61.8 times greater for PEP-PCR and 220.5 times greater than the unamplified controls for DOP-PCR.
  
  
  CONCLUSIONS
  Of the amplification methodologies examined in this paper, the multiple displacement amplification products generated the least bias, and produced significantly higher yields of amplified DNA.
authors.affiliation: 454 Life Sciences, 20 Commercial Street, Branford CT 06405, USA. robert.pinard@historx.com
authors.forename: Robert
authors.initials: R
authors.lastname: Pinard
authors.forename: Alex
authors.initials: A
authors.lastname: de Winter
authors.forename: Gary J
authors.initials: GJ
authors.lastname: Sarkis
authors.forename: Mark B
authors.initials: MB
authors.lastname: Gerstein
authors.forename: Karrie R
authors.initials: KR
authors.lastname: Tartaro
authors.forename: Ramona N
authors.initials: RN
authors.lastname: Plant
authors.forename: Michael
authors.initials: M
authors.lastname: Egholm
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Rothberg
authors.forename: John H
authors.initials: JH
authors.lastname: Leamon
mesh_terms: D015982:Bias; D016123:Campylobacter jejuni / Q000235:genetics; D002876:Chromosomes, Bacterial; D015342:DNA Probes; D016680:Genome, Bacterial; D023281:Genomics / Q000379:methods* / Q000706:statistics & numerical data; D006217:Halobacterium / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques; D017422:Sequence Analysis, DNA / Q000379:methods*; D018709:Statistics, Nonparametric
chemical_list: D015342:DNA Probes