UTILITY OF NEXT-GENERATION SEQUENCING FOR INTEGRATED MOLECULAR PROFILING OF PRIMARY MYELOFIBROSIS USING ION TORRENT PGM SYSTEM
(Abstract release date: 05/21/15)
EHA Library. Shivarov V. 06/12/15; 102862; PB1917
Disclosure(s): Sofiamed University HospitalLaboratory of Clinical Immunology
Dr. Velizar Shivarov
Contributions
Contributions
Abstract
Abstract: PB1917
Type: Publication Only
Background
In the last decade a number of genes have been reported to be recurrently associated with primary myelofiborsis (PMF). While some mutations such as JAK2 V617F and MPL exon 10 mutations are easily detectable by conventional molecular genetics methods other mutations are more difficult for screening because of lower frequency and being scattered along large genomic ranges. On the other hand, newly developed approaches for next-generation sequencing (NGS) provide an affordable solution for targeted multiplex resequencing of up-to several hundreds of amplicons.
Aims
Here, we aimed at the development and validation of a novel custom panel for targeted resequencing of PMF samples using the Ion PGMTM System (Ion Torrent, USA).
Methods
We designed a pool of 424 primers for amplification of 212 amplicons covering 99.46% of the exonic regions of 9 human genes as follows: ASXL1, EZH2, CALR, RUNX1, SETBP1, SF3B1, SRSF2, TET2 and U2AF1. Initial testing of the panel performance was done on Ion PGMTM machine using PGMTM 316 v2 chips on 8 DNA samples from PMF patients (6 males, 2 females, median age 61.5 years). Sequences alignment, variants calling and annotation were performed using Ion Reporter software.
Results
Summary
This small proof-of-concept study confirms the feasibility of Ion Torrent systems for resequencing of clinically relevant mutations in myeloid malignancies such as PMF. It can be particularly useful in cases without the most frequent clonal markers in PMF such as JAK2, MPL and CALR mutations. Acknowledgements: The authors are thankful to Dr. Nina Petkova and Dr. Evgenii Hadzhiev for providing the clinical samples as part of the project ID_09_157 (National Science Fund, Bulgaria).
Keyword(s): Molecular markers, Mutation analysis, Myelofibrosis
Session topic: Publication Only
Type: Publication Only
Background
In the last decade a number of genes have been reported to be recurrently associated with primary myelofiborsis (PMF). While some mutations such as JAK2 V617F and MPL exon 10 mutations are easily detectable by conventional molecular genetics methods other mutations are more difficult for screening because of lower frequency and being scattered along large genomic ranges. On the other hand, newly developed approaches for next-generation sequencing (NGS) provide an affordable solution for targeted multiplex resequencing of up-to several hundreds of amplicons.
Aims
Here, we aimed at the development and validation of a novel custom panel for targeted resequencing of PMF samples using the Ion PGMTM System (Ion Torrent, USA).
Methods
We designed a pool of 424 primers for amplification of 212 amplicons covering 99.46% of the exonic regions of 9 human genes as follows: ASXL1, EZH2, CALR, RUNX1, SETBP1, SF3B1, SRSF2, TET2 and U2AF1. Initial testing of the panel performance was done on Ion PGMTM machine using PGMTM 316 v2 chips on 8 DNA samples from PMF patients (6 males, 2 females, median age 61.5 years). Sequences alignment, variants calling and annotation were performed using Ion Reporter software.
Results
We identified a total of 22 nonsynonymos somatic coding variants in 7 out of 8 samples. The most frequently mutated gene was TET2 (6 mutated samples out of 8). Two patients had mutations in SRSF2, and CALR, EZH2 and SETBP1 were found mutated in one sample each. No mutations in RUNX1, SF3B1 and U2AF1 were found. Overall, mutations rate was consistent with previous reports on PMF molecular profiling.
Summary
This small proof-of-concept study confirms the feasibility of Ion Torrent systems for resequencing of clinically relevant mutations in myeloid malignancies such as PMF. It can be particularly useful in cases without the most frequent clonal markers in PMF such as JAK2, MPL and CALR mutations. Acknowledgements: The authors are thankful to Dr. Nina Petkova and Dr. Evgenii Hadzhiev for providing the clinical samples as part of the project ID_09_157 (National Science Fund, Bulgaria).
Keyword(s): Molecular markers, Mutation analysis, Myelofibrosis
Session topic: Publication Only
Abstract: PB1917
Type: Publication Only
Background
In the last decade a number of genes have been reported to be recurrently associated with primary myelofiborsis (PMF). While some mutations such as JAK2 V617F and MPL exon 10 mutations are easily detectable by conventional molecular genetics methods other mutations are more difficult for screening because of lower frequency and being scattered along large genomic ranges. On the other hand, newly developed approaches for next-generation sequencing (NGS) provide an affordable solution for targeted multiplex resequencing of up-to several hundreds of amplicons.
Aims
Here, we aimed at the development and validation of a novel custom panel for targeted resequencing of PMF samples using the Ion PGMTM System (Ion Torrent, USA).
Methods
We designed a pool of 424 primers for amplification of 212 amplicons covering 99.46% of the exonic regions of 9 human genes as follows: ASXL1, EZH2, CALR, RUNX1, SETBP1, SF3B1, SRSF2, TET2 and U2AF1. Initial testing of the panel performance was done on Ion PGMTM machine using PGMTM 316 v2 chips on 8 DNA samples from PMF patients (6 males, 2 females, median age 61.5 years). Sequences alignment, variants calling and annotation were performed using Ion Reporter software.
Results
Summary
This small proof-of-concept study confirms the feasibility of Ion Torrent systems for resequencing of clinically relevant mutations in myeloid malignancies such as PMF. It can be particularly useful in cases without the most frequent clonal markers in PMF such as JAK2, MPL and CALR mutations. Acknowledgements: The authors are thankful to Dr. Nina Petkova and Dr. Evgenii Hadzhiev for providing the clinical samples as part of the project ID_09_157 (National Science Fund, Bulgaria).
Keyword(s): Molecular markers, Mutation analysis, Myelofibrosis
Session topic: Publication Only
Type: Publication Only
Background
In the last decade a number of genes have been reported to be recurrently associated with primary myelofiborsis (PMF). While some mutations such as JAK2 V617F and MPL exon 10 mutations are easily detectable by conventional molecular genetics methods other mutations are more difficult for screening because of lower frequency and being scattered along large genomic ranges. On the other hand, newly developed approaches for next-generation sequencing (NGS) provide an affordable solution for targeted multiplex resequencing of up-to several hundreds of amplicons.
Aims
Here, we aimed at the development and validation of a novel custom panel for targeted resequencing of PMF samples using the Ion PGMTM System (Ion Torrent, USA).
Methods
We designed a pool of 424 primers for amplification of 212 amplicons covering 99.46% of the exonic regions of 9 human genes as follows: ASXL1, EZH2, CALR, RUNX1, SETBP1, SF3B1, SRSF2, TET2 and U2AF1. Initial testing of the panel performance was done on Ion PGMTM machine using PGMTM 316 v2 chips on 8 DNA samples from PMF patients (6 males, 2 females, median age 61.5 years). Sequences alignment, variants calling and annotation were performed using Ion Reporter software.
Results
We identified a total of 22 nonsynonymos somatic coding variants in 7 out of 8 samples. The most frequently mutated gene was TET2 (6 mutated samples out of 8). Two patients had mutations in SRSF2, and CALR, EZH2 and SETBP1 were found mutated in one sample each. No mutations in RUNX1, SF3B1 and U2AF1 were found. Overall, mutations rate was consistent with previous reports on PMF molecular profiling.
Summary
This small proof-of-concept study confirms the feasibility of Ion Torrent systems for resequencing of clinically relevant mutations in myeloid malignancies such as PMF. It can be particularly useful in cases without the most frequent clonal markers in PMF such as JAK2, MPL and CALR mutations. Acknowledgements: The authors are thankful to Dr. Nina Petkova and Dr. Evgenii Hadzhiev for providing the clinical samples as part of the project ID_09_157 (National Science Fund, Bulgaria).
Keyword(s): Molecular markers, Mutation analysis, Myelofibrosis
Session topic: Publication Only
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