Systematic bias in high-throughput sequencing data and its correction by BEADS

Nucleic Acids Res. 2011 Aug;39(15):e103. doi: 10.1093/nar/gkr425. Epub 2011 Jun 6.

Abstract

Genomic sequences obtained through high-throughput sequencing are not uniformly distributed across the genome. For example, sequencing data of total genomic DNA show significant, yet unexpected enrichments on promoters and exons. This systematic bias is a particular problem for techniques such as chromatin immunoprecipitation, where the signal for a target factor is plotted across genomic features. We have focused on data obtained from Illumina's Genome Analyser platform, where at least three factors contribute to sequence bias: GC content, mappability of sequencing reads, and regional biases that might be generated by local structure. We show that relying on input control as a normalizer is not generally appropriate due to sample to sample variation in bias. To correct sequence bias, we present BEADS (bias elimination algorithm for deep sequencing), a simple three-step normalization scheme that successfully unmasks real binding patterns in ChIP-seq data. We suggest that this procedure be done routinely prior to data interpretation and downstream analyses.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algorithms
  • Animals
  • Base Composition
  • Caenorhabditis elegans / genetics
  • Chromatin Immunoprecipitation*
  • DNA, Helminth / chemistry
  • High-Throughput Nucleotide Sequencing / methods*
  • Sequence Analysis, DNA / methods*

Substances

  • DNA, Helminth