A bumpy ride on the diagnostic bench of massive parallel sequencing, the case of the mitochondrial genome.
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. 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.
Research Group Reproduction and Genetics (REGE), Vrije Universiteit Brussel (VUB), Brussels, Belgium.
Kim
K
Vancampenhout
Center for Medical Genetics, UZ Brussel, Vrije Universiteit Brussel (VUB), Brussels, Belgium.
Ben
B
Caljon
Research Group Reproduction and Genetics (REGE), Vrije Universiteit Brussel (VUB), Brussels, Belgium.
Claudia
C
Spits
Research Group Reproduction and Genetics (REGE), Vrije Universiteit Brussel (VUB), Brussels, Belgium; Center for Medical Genetics, UZ Brussel, Vrije Universiteit Brussel (VUB), Brussels, Belgium.
Katrien
K
Stouffs
Department of Pediatric Neurology, UZ Brussel, Vrije Universiteit Brussel (VUB), Brussels, Belgium.
An
A
Jonckheere
Research Group Reproduction and Genetics (REGE), Vrije Universiteit Brussel (VUB), Brussels, Belgium; Department of Pediatric Neurology, UZ Brussel, Vrije Universiteit Brussel (VUB), Brussels, Belgium.
Linda
L
De Meirleir
Research Group Reproduction and Genetics (REGE), Vrije Universiteit Brussel (VUB), Brussels, Belgium; Center for Medical Genetics, UZ Brussel, Vrije Universiteit Brussel (VUB), Brussels, Belgium.
Willy
W
Lissens
Department of Pediatrics, Division of Pediatric Neurology and Metabolism, University Hospital Ghent, Ghent University, Ghent, Belgium.
Arnaud
A
Vanlander
Department of Pediatrics, Division of Pediatric Neurology and Metabolism, University Hospital Ghent, Ghent University, Ghent, Belgium.
Joél
J
Smet
Department of Pediatrics, Division of Pediatric Neurology and Metabolism, University Hospital Ghent, Ghent University, Ghent, Belgium.
Boel
B
De Paepe
Department of Pediatrics, Division of Pediatric Neurology and Metabolism, University Hospital Ghent, Ghent University, Ghent, Belgium.
Rudy
R
Van Coster
Research Group Reproduction and Genetics (REGE), Vrije Universiteit Brussel (VUB), Brussels, Belgium; Center for Medical Genetics, UZ Brussel, Vrije Universiteit Brussel (VUB), Brussels, Belgium.
Sara
S
Seneca
D004272:DNA, Mitochondrial / Q000235:genetics; D015723:Gene Library; D054629:Genome, Mitochondrial; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D010865:Pilot Projects; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*
D004272:DNA, Mitochondrial