Detection of mutations in myeloid malignancies through paired-sample analysis of Microdroplet-PCR deep sequencing data Journal Article


Authors: Cheng, D. T.; Cheng, J.; Mitchell, T. N.; Syed, A.; Zehir, A.; Mensah, N. Y. T.; Oultache, A.; Nafa, K.; Levine, R. L.; Arcila, M. E.; Berger, M. F.; Hedvat, C. V.
Article Title: Detection of mutations in myeloid malignancies through paired-sample analysis of Microdroplet-PCR deep sequencing data
Abstract: Amplicon-based methods for targeted resequencing of cancer genes have gained traction in the clinic as a strategy for molecular diagnostic testing. An 847-amplicon panel was designed with the RainDance DeepSeq system, covering most exons of 28 genes relevant to acute myeloid leukemia and myeloproliferative neoplasms. We developed a paired-sample analysis pipeline for variant calling and sought to assess its sensitivity and specificity relative to a set of samples with previously identified mutations. Thirty samples with known mutations in JAK2, NPM1, DNMT3A, MPL, IDH1, IDH2, CEBPA, and FLT3, were profiled and sequenced to high depth. Variant calling using an unmatched Hapmap DNA control removed a substantial number of artifactual calls regardless of algorithm used or variant class. The removed calls were nonunique, had lower variant frequencies, and tended to recur in multiple unrelated samples. Analysis of sample replicates revealed that reproducible calls had distinctly higher variant allele depths and frequencies compared to nonreproducible calls. On the basis of these differences, filters on variant frequency were chosen to select for reproducible calls. The analysis pipeline successfully retrieved the associated known variant in all tested samples and uncovered additional mutations in some samples corresponding to well-characterized hotspot mutations in acute myeloid leukemia. We have developed a paired-sample analysis pipeline capable of robust identification of mutations from microdroplet-PCR sequencing data with high sensitivity and specificity. © 2014 American Society for Investigative Pathology.
Journal Title: Journal of Molecular Diagnostics
Volume: 16
Issue: 5
ISSN: 1525-1578
Publisher: Elsevier Science, Inc.  
Date Published: 2014-09-01
Start Page: 504
End Page: 518
Language: English
DOI: 10.1016/j.jmoldx.2014.05.006
PROVIDER: scopus
PUBMED: 25017477
PMCID: PMC5707185
DOI/URL:
Notes: Export Date: 2 September 2014 -- CODEN: JMDIF -- Source: Scopus
Altmetric Score
MSK Authors
  1. Khedoudja Nafa
    184 Nafa
  2. Cyrus Hedvat
    123 Hedvat
  3. Ross Levine
    489 Levine
  4. Ahmet Zehir
    153 Zehir
  5. Michael Forman Berger
    386 Berger
  6. Maria Eugenia Arcila
    343 Arcila
  7. Nana Yaa Takyiwaa Mensah
    7 Mensah
  8. Janice Cheng
    2 Cheng
  9. Donavan Tai Suan Cheng
    51 Cheng
  10. Aijazuddin Syed
    20 Syed