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 language | English |
|---|---|
| Pages (from-to) | 2967-2974 |
| Number of pages | 8 |
| Journal | Molecular Medicine Reports |
| Volume | 14 |
| Issue number | 4 |
| DOIs | |
| Publication status | Published - Oct 1 2016 |
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Keywords
- AmpliSeq
- Chimerism
- Ion Torrent
- Next generation sequencing
- Quantification
- SNP
ASJC Scopus subject areas
- Biochemistry
- Molecular Medicine
- Molecular Biology
- Oncology
- Genetics
- Cancer Research
Cite this
A technical application of quantitative next generation sequencing for chimerism evaluation. / Aloisio, Michelangelo; Licastro, Danilo; Caenazzo, Luciana; Torboli, Valentina; D'Eustacchio, Angela; Severini, Giovanni Maria; Athanasakis, Emmanouil.
In: Molecular Medicine Reports, Vol. 14, No. 4, 01.10.2016, p. 2967-2974.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - A technical application of quantitative next generation sequencing for chimerism evaluation
AU - Aloisio, Michelangelo
AU - Licastro, Danilo
AU - Caenazzo, Luciana
AU - Torboli, Valentina
AU - D'Eustacchio, Angela
AU - Severini, Giovanni Maria
AU - Athanasakis, Emmanouil
PY - 2016/10/1
Y1 - 2016/10/1
N2 - 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.
AB - 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.
KW - AmpliSeq
KW - Chimerism
KW - Ion Torrent
KW - Next generation sequencing
KW - Quantification
KW - SNP
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U2 - 10.3892/mmr.2016.5593
DO - 10.3892/mmr.2016.5593
M3 - Article
AN - SCOPUS:84988498998
VL - 14
SP - 2967
EP - 2974
JO - Molecular Medicine Reports
JF - Molecular Medicine Reports
SN - 1791-2997
IS - 4
ER -