A bumpy ride on the diagnostic bench of massive parallel sequencing, the case of the mitochondrial genome

Kim Vancampenhout, Ben Caljon, Claudia Spits, Katrien Stouffs, An Jonckheere, Linda De Meirleir, Willy Lissens, A. Vanlander, Joél Smet, Boel De Paepe, Rudy Van Coster, Sara Seneca

Research output: Contribution to journalArticlepeer-review

14 Citations (Scopus)

Abstract

The advent of massive parallel sequencing (MPS) has revolutionized the field of human molecular genetics, including the diagnostic study of mitochondrial (mt) DNA dysfunction. The analysis of the complete mitochondrial genome using MPS platforms is now common and will soon outrun conventional sequencing. However, the development of a robust and reliable protocol is rather challenging. A previous pilot study for the re-sequencing of human mtDNA revealed an uneven coverage, affecting predominantly part of the plus strand. This might lead to unreliable data of the targeted mitochondrial genomes, making the system less suitable for clinical diagnosis for this specific application. In an attempt to address this problem, we undertook a comparative study of standard and modified protocols for the Ion Torrent PGM system. We could not improve strand representation by altering the recommended shearing methodology of the standard workflow or omitting the DNA polymerase amplification step from the library construction process. However, we were able to associate coverage bias of the plus strand with a specific sequence motif. Additionally, we compared coverage and variant calling across technologies. The same samples were also sequenced on a MiSeq device which showed that coverage and heteroplasmic variant calling were much improved.
Original languageEnglish
Article numbere112950
Number of pages9
JournalPLoS ONE
Volume9
Issue number11
Publication statusPublished - 2014

Keywords

  • mtDNA
  • NGS
  • MPS
  • Ion Torrent PGM

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