A technical application of quantitative next generation sequencing for chimerism evaluation

Michelangelo Aloisio, Danilo Licastro, Luciana Caenazzo, Valentina Torboli, Angela D'Eustacchio, Giovanni Maria Severini, Emmanouil Athanasakis

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Pages (from-to)2967-2974
Number of pages8
JournalMolecular Medicine Reports
Volume14
Issue number4
DOIs
Publication statusPublished - Oct 1 2016

Keywords

  • AmpliSeq
  • Chimerism
  • Ion Torrent
  • Next generation sequencing
  • Quantification
  • SNP

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Oncology
  • Genetics
  • Cancer Research

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