Reproducibility of Illumina platform deep sequencing errors allows accurate determination of DNA barcodes in cells

BMC Bioinformatics. 2016 Apr 2;17:151. doi: 10.1186/s12859-016-0999-4.

Abstract

Background: Next generation sequencing (NGS) of amplified DNA is a powerful tool to describe genetic heterogeneity within cell populations that can both be used to investigate the clonal structure of cell populations and to perform genetic lineage tracing. For applications in which both abundant and rare sequences are biologically relevant, the relatively high error rate of NGS techniques complicates data analysis, as it is difficult to distinguish rare true sequences from spurious sequences that are generated by PCR or sequencing errors. This issue, for instance, applies to cellular barcoding strategies that aim to follow the amount and type of offspring of single cells, by supplying these with unique heritable DNA tags.

Results: Here, we use genetic barcoding data from the Illumina HiSeq platform to show that straightforward read threshold-based filtering of data is typically insufficient to filter out spurious barcodes. Importantly, we demonstrate that specific sequencing errors occur at an approximately constant rate across different samples that are sequenced in parallel. We exploit this observation by developing a novel approach to filter out spurious sequences.

Conclusions: Application of our new method demonstrates its value in the identification of true sequences amongst spurious sequences in biological data sets.

Keywords: Cellular barcoding; Illumina; Lineage tracing; Next generation sequencing; PCR error; Sequencing error.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • DNA / analysis*
  • DNA / chemistry
  • DNA Barcoding, Taxonomic*
  • High-Throughput Nucleotide Sequencing*
  • Mice
  • Polymerase Chain Reaction
  • Sequence Analysis, DNA
  • Stem Cells / cytology
  • Stem Cells / metabolism

Substances

  • DNA