Streamlined ion torrent PGM-based diagnostics: BRCA1 and BRCA2 genes as a model.
To meet challenges in terms of throughput and turnaround time, many diagnostic laboratories are shifting from Sanger sequencing to higher throughput next-generation sequencing (NGS) platforms. Bearing in mind that the performance and quality criteria expected from NGS in diagnostic or research settings are strikingly different, we have developed an Ion Torrent's PGM-based routine diagnostic procedure for BRCA1/2 sequencing. The procedure was first tested on a training set of 62 control samples, and then blindly validated on 77 samples in parallel with our routine technique. The training set was composed of difficult cases, for example, insertions and/or deletions of various sizes, large-scale rearrangements and, obviously, mutations occurring in homopolymer regions. We also compared two bioinformatic solutions in this diagnostic context, an in-house academic pipeline and the commercially available NextGene software (Softgenetics). NextGene analysis provided higher sensitivity, as four previously undetected single-nucleotide variations were found. Regarding specificity, an average of 1.5 confirmatory Sanger sequencings per patient was needed for complete BRCA1/2 screening. Large-scale rearrangements were identified by two distinct analyses, that is, bioinformatics and fragment analysis with electrophoresis profile comparison. Turnaround time was enhanced, as a series of 30 patients were sequenced by one technician, making the results available for the clinician in 10 working days following blood sampling. BRCA1/2 genes are a good model, representative of the difficulties commonly encountered in diagnostic settings, which is why we believe our findings are of interest for the whole community, and the pipeline described can be adapted by any user of PGM for diagnostic purposes.
1] Service de Génétique Oncologique, Institut Curie, Paris, France [2] INSERM U830, Centre de Recherche de l'Institut Curie, Paris, France.
Julien
J
Tarabeux
INSERM U900, Mines Paris Tech, Centre de Recherche de l'Institut Curie, Paris, France.
Bruno
B
Zeitouni
Service de Génétique Oncologique, Institut Curie, Paris, France.
Virginie
V
Moncoutier
Service de Génétique Oncologique, Institut Curie, Paris, France.
Henrique
H
Tenreiro
Service de Génétique Oncologique, Institut Curie, Paris, France.
Khadija
K
Abidallah
INSERM U900, Mines Paris Tech, Centre de Recherche de l'Institut Curie, Paris, France.
Séverine
S
Lair
Next Generation Sequencing Platform, Centre de Recherche de l'Institut Curie, Paris, France.
Patricia
P
Legoix-Né
Next Generation Sequencing Platform, Centre de Recherche de l'Institut Curie, Paris, France.
Quentin
Q
Leroy
Service de Génétique Oncologique, Institut Curie, Paris, France.
Etienne
E
Rouleau
1] Service de Génétique Oncologique, Institut Curie, Paris, France [2] INSERM U830, Centre de Recherche de l'Institut Curie, Paris, France.
Lisa
L
Golmard
INSERM U900, Mines Paris Tech, Centre de Recherche de l'Institut Curie, Paris, France.
Emmanuel
E
Barillot
1] Service de Génétique Oncologique, Institut Curie, Paris, France [2] INSERM U830, Centre de Recherche de l'Institut Curie, Paris, France.
Marc-Henri
MH
Stern
Next Generation Sequencing Platform, Centre de Recherche de l'Institut Curie, Paris, France.
Thomas
T
Rio-Frio
1] Service de Génétique Oncologique, Institut Curie, Paris, France [2] INSERM U830, Centre de Recherche de l'Institut Curie, Paris, France [3] Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
Dominique
D
Stoppa-Lyonnet
1] Service de Génétique Oncologique, Institut Curie, Paris, France [2] INSERM U830, Centre de Recherche de l'Institut Curie, Paris, France [3] Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
Claude
C
Houdayer
D019313:BRCA1 Protein / Q000235:genetics*; D024682:BRCA2 Protein / Q000235:genetics*; D001943:Breast Neoplasms / Q000175:diagnosis / Q000235:genetics; D019295:Computational Biology; D005260:Female; D015321:Gene Rearrangement; D005820:Genetic Testing; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D010051:Ovarian Neoplasms / Q000175:diagnosis / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D012984:Software
D019313:BRCA1 Protein; C492913:BRCA1 protein, human; D024682:BRCA2 Protein; C551750:BRCA2 protein, human