Dealing with Pseudogenes in Molecular Diagnostics in the Next Generation Sequencing Era

Methods Mol Biol. 2021;2324:363-381. doi: 10.1007/978-1-0716-1503-4_22.


Presence of pseudogenes is a dreadful issue in next generation sequencing (NGS), because their contamination can interfere with the detection of variants in the genuine gene and generate false positive and false negative variants.In this chapter we focus on issues related to the application of NGS strategies for analysis of genes with pseudogenes in a clinical setting. The degree to which a pseudogene impacts the ability to accurately detect and map variants in its parent gene depends on the degree of similarity (homology) with the parent gene itself. Hereby, target enrichment and mapping strategies are crucial factors to avoid "contaminating" pseudogene sequences. For target enrichment, we describe advantages and disadvantages of PCR- and capture-based strategies. For mapping strategies, we discuss crucial parameters that need to be considered to accurately distinguish sequences of functional genes from pseudogenic sequences. Finally, we discuss some examples of genes associated with Mendelian disorders, for which interesting NGS approaches are described to avoid interference with pseudogene sequences.

Keywords: Data analysis in NGS; Molecular diagnostics; Next generation sequencing (NGS); Pseudogenes of genes associated with monogenic disorders; Target enrichment in NGS.

Publication types

  • Review

MeSH terms

  • Base Sequence
  • Data Analysis
  • Electrophoresis, Capillary / methods
  • Genes, BRCA1
  • Genes, Neurofibromatosis 1
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • INDEL Mutation
  • Intercellular Signaling Peptides and Proteins / genetics
  • Mismatch Repair Endonuclease PMS2 / genetics
  • Molecular Diagnostic Techniques*
  • Multiplex Polymerase Chain Reaction / methods
  • Oligonucleotide Probes
  • Polymorphism, Single Nucleotide
  • Pseudogenes*
  • Reverse Transcriptase Polymerase Chain Reaction / methods
  • TRPP Cation Channels / genetics


  • Intercellular Signaling Peptides and Proteins
  • Oligonucleotide Probes
  • STRC protein, human
  • TRPP Cation Channels
  • polycystic kidney disease 1 protein
  • polycystic kidney disease 2 protein
  • PMS2 protein, human
  • Mismatch Repair Endonuclease PMS2