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.

Patient-derived xenotransplants can recapitulate the genetic driver landscape of acute leukemias

“…This finding either suggests that most of the rare clones (SVAF <10%) change their share in the bulk leukemia, or reflects a high technical standard deviation (SD) of the detection of low frequency events. To assess sensitivity and accuracy of FoundationOne Heme test we sequenced amplicons of 30 genes 15 from 7 PDX samples, which resulted in detection of all 17 SVAF. Plotted SVAF from FoundationOne Heme and 30-gene amplicons demonstrated highly correlated (R 2 =0.802, p<10 -4 ) but not uniform data, suggesting that the SVAF obtained form a single method should be considered to be used with a wider SD, than calculated using the variance and depth-coverage from the single method, figure 1C.…”

“…All captured libraries were sequenced on HiSeq2500, Illumina 14 . For amplicon-sequencing gDNA was isolated from 7 available and not related PDX samples, selected genomic regions were amplified using microdroplet-PCR followed by illumina sequencing 15 . Excluding PE100 reads that align to mouse genome (mm9) in PDX samples resulted in loss <0.6% reads, which was considered not to affect VAF and therefore disregarded 16 .…”

The oncocytic subtype is genetically distinct from other pancreatic intraductal papillary mucinous neoplasm subtypes

“…Deep coverage, targeted next-generation sequencing was then performed on a panel of 300 genes, including KRAS, GNAS , and RNF43 , listed in the supporting information (Supplementary Information File 1), known to undergo somatic genomic alterations in cancer, as previously described. 37,38 Briefly, massively parallel sequencing libraries (Kapa Biosystems, New England Biolabs) that contain barcoded universal primers were generated from 115 to 250 ng genomic DNA from the tumor material and matched normal tissue. After library amplification and DNA quantification, equimolar pools were generated consisting of up to 24 barcoded libraries.…”

Allogeneic Hematopoietic Stem Cell Transplantation with Myeloablative Conditioning Is Associated with Favorable Outcomes in Mixed Phenotype Acute Leukemia

“…Bone marrow from 8 cases was sequenced using a NGS platform that sequences DNA from 405 genes and RNA from 265 genes of known oncogenic drivers in hematologic malignancies including AML and ALL, sarcomas and pediatric cancer 12 . Additionally, 8 MPAL were sequenced using an institutional NGS platform, which targets 28 genes recurrently mutated in myeloid neoplasms 13 . Minimal residual disease (MRD) at the time of HSCT was assessed using various methods including 10-colour flow cytometry (n=9), quantitative PCR for BCR ABL (n=5), FISH for patient specific leukemia-defining cytogenetic alteration (n=3), presence of IHG or TCR rearrangement (n=3).…”