title: A technical application of quantitative next generation sequencing for chimerism evaluation.
abstract: At present, the most common genetic diagnostic method for chimerism evaluation following hematopoietic stem cell transplantation is microsatellite analysis by capillary electrophoresis. The main objective was to establish, through repeated analysis over time, if a complete chimerism was present, or if the mixed chimerism was stable, increasing or decreasing over time. Considering the recent introduction of next generation sequencing (NGS) in clinical diagnostics, a detailed study evaluating an NGS protocol was conducted, coupled with a custom bioinformatics pipeline, for chimerism quantification. Based on the technology of Ion AmpliSeq, a 44‑amplicon custom chimerism panel was designed, and a custom bioinformatics pipeline dedicated to the genotyping and quantification of NGS data was coded. The custom chimerism panel allowed identification of an average of 16 informative recipient alleles. The limit of detection of the protocol was fixed at 1% due to the NGS background (<1%). The protocol followed the standard Ion AmpliSeq library preparation and Ion Torrent Personal Genome Machine guidelines. Overall, the present study added to the scientific literature, identifying novel technical details for a possible future application of NGS for chimerism quantification.
authors: Department of Life Sciences, University of Trieste, I‑34127 Trieste, Italy.
authors: Michelangelo
authors: M
authors: Aloisio
authors: Cluster in Biomedicine, CBM S.c.r.l., Bioinformatic Services, Area Science Park, I‑34149 Basovizza, Italy.
authors: Danilo
authors: D
authors: Licastro
authors: Department of Molecular Medicine, University of Padova, I‑35121 Padova, Italy.
authors: Luciana
authors: L
authors: Caenazzo
authors: Department of Life Sciences, University of Trieste, I‑34127 Trieste, Italy.
authors: Valentina
authors: V
authors: Torboli
authors: Department of Advanced Diagnostic and Clinical Trials, Institute for Maternal and Child Health, IRCCS 'Burlo Garofolo', I‑34137 Trieste, Italy.
authors: Angela
authors: A
authors: D'Eustacchio
authors: Department of Advanced Diagnostic and Clinical Trials, Institute for Maternal and Child Health, IRCCS 'Burlo Garofolo', I‑34137 Trieste, Italy.
authors: Giovanni Maria
authors: GM
authors: Severini
authors: Department of Advanced Diagnostic and Clinical Trials, Institute for Maternal and Child Health, IRCCS 'Burlo Garofolo', I‑34137 Trieste, Italy.
authors: Emmanouil
authors: E
authors: Athanasakis
mesh_terms: D000328:Adult; D046528:Chimerism; D005260:Female; D023281:Genomics / Q000379:methods; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008297:Male; D018895:Microsatellite Repeats; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis; D017422:Sequence Analysis, DNA / Q000379:methods; D055815:Young Adult