title: Impact of normalization methods on high-throughput screening data with high hit rates and drug testing with dose-response data.
abstract: BACKGROUND
  Most data analysis tools for high-throughput screening (HTS) seek to uncover interesting hits for further analysis. They typically assume a low hit rate per plate. Hit rates can be dramatically higher in secondary screening, RNAi screening and in drug sensitivity testing using biologically active drugs. In particular, drug sensitivity testing on primary cells is often based on dose-response experiments, which pose a more stringent requirement for data quality and for intra- and inter-plate variation. Here, we compared common plate normalization and noise-reduction methods, including the B-score and the Loess a local polynomial fit method under high hit-rate scenarios of drug sensitivity testing. We generated simulated 384-well plate HTS datasets, each with 71 plates having a range of 20 (5%) to 160 (42%) hits per plate, with controls placed either at the edge of the plates or in a scattered configuration.
  
  
  RESULTS
  We identified 20% (77/384) as the critical hit-rate after which the normalizations started to perform poorly. Results from real drug testing experiments supported this estimation. In particular, the B-score resulted in incorrect normalization of high hit-rate plates, leading to poor data quality, which could be attributed to its dependency on the median polish algorithm. We conclude that a combination of a scattered layout of controls per plate and normalization using a polynomial least squares fit method, such as Loess helps to reduce column, row and edge effects in HTS experiments with high hit-rates and is optimal for generating accurate dose-response curves.
  
  
  BACKGROUND
  john.mpindi@helsinki.fi.
  
  
  METHODS
  Supplementary information: R code and Supplementary data are available at Bioinformatics online.
authors.affiliation: University of Helsinki, Institute for Molecular Medicine, Tukholmankatu 8, FI-00290, Helsinki, Finland.
authors.forename: John-Patrick
authors.initials: JP
authors.lastname: Mpindi
authors.affiliation: University of Helsinki, Institute for Molecular Medicine, Tukholmankatu 8, FI-00290, Helsinki, Finland.
authors.forename: Potdar
authors.initials: P
authors.lastname: Swapnil
authors.affiliation: University of Helsinki, Institute for Molecular Medicine, Tukholmankatu 8, FI-00290, Helsinki, Finland.
authors.forename: Bychkov
authors.initials: B
authors.lastname: Dmitrii
authors.affiliation: University of Helsinki, Institute for Molecular Medicine, Tukholmankatu 8, FI-00290, Helsinki, Finland.
authors.forename: Saarela
authors.initials: S
authors.lastname: Jani
authors.affiliation: University of Helsinki, Institute for Molecular Medicine, Tukholmankatu 8, FI-00290, Helsinki, Finland.
authors.forename: Khalid
authors.initials: K
authors.lastname: Saeed
authors.affiliation: University of Helsinki, Institute for Molecular Medicine, Tukholmankatu 8, FI-00290, Helsinki, Finland.
authors.forename: Krister
authors.initials: K
authors.lastname: Wennerberg
authors.affiliation: University of Helsinki, Institute for Molecular Medicine, Tukholmankatu 8, FI-00290, Helsinki, Finland.
authors.forename: Tero
authors.initials: T
authors.lastname: Aittokallio
authors.affiliation: University of Helsinki, Institute for Molecular Medicine, Tukholmankatu 8, FI-00290, Helsinki, Finland.
authors.forename: Päivi
authors.initials: P
authors.lastname: Östling
authors.affiliation: University of Helsinki, Institute for Molecular Medicine, Tukholmankatu 8, FI-00290, Helsinki, Finland.
authors.forename: Olli
authors.initials: O
authors.lastname: Kallioniemi
mesh_terms: D000465:Algorithms; D000970:Antineoplastic Agents / Q000494:pharmacology*; D003627:Data Interpretation, Statistical; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008297:Male; D016011:Normal Distribution; D011471:Prostatic Neoplasms / Q000188:drug therapy*; D014407:Tumor Cells, Cultured
chemical_list: D000970:Antineoplastic Agents