Abstract 5269: Minor Variant Finder: New software for detecting somatic mutations at low level in Sanger sequencing traces

2016 ◽  
Author(s):  
Edgar H. Schreiber ◽  
Harrison Leong ◽  
Stephanie J. Schneider ◽  
Marks Jeff ◽  
Wallace George ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Somatic Mutations ◽  
Low Level ◽  
Minor Variant

scholarly journals Using the Minor Variant Finder software to identify and quantify the allelic burden level of somatic mutations in oncohematologic diseases

2020 ◽  
Vol 15 (2) ◽  
pp. 85-91
Author(s):  
T. N. Subbotina ◽  
I. E. Maslyukova ◽  
A. A. Faleeva ◽  
P. A. Nikolaeva ◽  
A. S. Khazieva ◽  
...  
Keyword(s):  
Mutant Allele ◽  
Sanger Sequencing ◽  
Quantitative Assessment ◽  
Somatic Mutations ◽  
Myeloproliferative Neoplasms ◽  
Mutant Gene ◽  
Positive Sample ◽  
Allele Burden ◽  
Minor Variant ◽  
Allelic Burden

Background. There are problems related to both quantitative assessment of an allele burden level of a mutant gene and interpretation of results in DNA samples with the burden level of the mutant allele less than 15–20 %, when using Sanger sequencing for analyzing somatic mutations. Applied Biosystems (USA) has developed new software Minor Variant Finder, which allows determining mutations with the allele burden level from 5 %.The objective: to determine the allele burden level and identification of minor variants of somatic mutations in the ASXL1, JAK2 genes and BCR-ABL oncogene using Minor Variant Finder software in patients with myeloproliferative neoplasms.Materials and methods. The level of mutant allele burden for 15 patients with myeloproliferative neoplasms was determined by the identified mutations using the Minor Variant Finder software, after analysis of point somatic mutations in the ASXL1, JAK2 genes and BCR-ABL oncogene by Sanger sequencing.Results. The allele burden level in all 5 ASXL1-positive samples and BCR-ABL-positive sample was determined as higher than 20 % using the Minor Variant Finder software. The allele burden level in 2 cases was higher than 20 % and in 7 cases lower than 20 %, when we analyzed 9 JAK2-positive samples.Conclusion. Minor Variant Finder software can be used to estimate the allele burden level and to identify minor variants of somatic mutations in the ASXL, JAK2 and BCR-ABL genes.

scholarly journals Reducing the Number of Mismatches between Hairs and Buccal References When Analysing mtDNA Heteroplasmic Variation by Massively Parallel Sequencing

Genes ◽  
2020 ◽  
Vol 11 (11) ◽  
pp. 1355
Author(s):  
Kristiaan J. van der Gaag ◽  
Stijn Desmyter ◽  
Sophie Smit ◽  
Lourdes Prieto ◽  
Titia Sijen
Keyword(s):  
Sanger Sequencing ◽  
Nuclear Dna ◽  
Massively Parallel Sequencing ◽  
Detection Threshold ◽  
Hair Shaft ◽  
Massively Parallel ◽  
Sequencing Data ◽  
Parallel Sequencing ◽  
Low Level ◽  
Minor Variant

In forensics, mitochondrial DNA (mtDNA) analysis is foremost applied to rootless hairs often lacking detectable nuclear DNA. Sanger sequencing is the routine mtDNA method in most forensic laboratories, even though interpretation of mixed samples and heteroplasmic sites can be challenging. Individuals may hold cells with low-level heteroplasmy variants below the detection threshold and other cells where this minor variant is the major one. This difference may be interpreted as a mismatch between reference and evidentiary trace samples, such as buccal specimens and rootless hairs. Such mismatches may be solved by Massively Parallel Sequencing (MPS), allowing more sensitive quantitative analysis for mixed positions than Sanger. The mtDNA control region was analysed in buccal reference samples from 26 individuals and 475 corresponding hairs by MPS and compared to Sanger sequencing data generated on the same samples. With MPS, mixed contributions down to 3% were regarded, leading to a substantial increase in the frequency of heteroplasmy. Our results demonstrate that previously reported mismatches between buccal reference and hair shaft samples by Sanger are detected as low-level heteroplasmy by MPS. A detailed overview of buccal and hair heteroplasmy is provided and implications for MPS-based mtDNA analysis in the context of forensic cases are discussed.

New software for detecting somatic mutations at low level in Sanger sequencing traces

2016 ◽  
Vol 61 ◽  
pp. S16
Author(s):  
E. Schreiber ◽  
H. Leong ◽  
S. Berosik ◽  
S. Schneider ◽  
J. Marks ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Somatic Mutations ◽  
Low Level

Rare Germline GLMN Variants Identified from Blue Rubber Bleb Nevus Syndrome Might Impact mTOR Signaling

2020 ◽  
Vol 22 (10) ◽  
pp. 675-682 ◽  
Author(s):  
Jie Yin ◽  
Zhongping Qin ◽  
Kai Wu ◽  
Yufei Zhu ◽  
Landian Hu ◽  
...  
Keyword(s):  
Gastrointestinal Tract ◽  
Sanger Sequencing ◽  
Somatic Mutations ◽  
Venous Malformation ◽  
Underlying Disease ◽  
Mtor Signaling ◽  
Functional Effect ◽  
Germline Variants ◽  
Whole Exome

Backgrounds and Objective: Blue rubber bleb nevus syndrome (BRBN) or Bean syndrome is a rare Venous Malformation (VM)-associated disorder, which mostly affects the skin and gastrointestinal tract in early childhood. Somatic mutations in TEK have been identified from BRBN patients; however, the etiology of TEK mutation-negative patients of BRBN need further investigation. Method: Two unrelated sporadic BRBNs and one sporadic VM were firstly screened for any rare nonsilent mutation in TEK by Sanger sequencing and subsequently applied to whole-exome sequencing to identify underlying disease causative variants. Overexpression assay and immunoblotting were used to evaluate the functional effect of the candidate disease causative variants. Results: In the VM case, we identified the known causative somatic mutation in the TEK gene c.2740C>T (p.Leu914Phe). In the BRBN patients, we identified two rare germline variants in GLMN gene c.761C>G (p.Pro254Arg) and c.1630G>T(p.Glu544*). The GLMN-P254R-expressing and GLMN-E544X-expressing HUVECs exhibited increased phosphorylation of mTOR-Ser-2448 in comparison with GLMN-WTexpressing HUVECs in vitro. Conclusion: Our results demonstrated that rare germline variants in GLMN might contribute to the pathogenesis of BRBN. Moreover, abnormal mTOR signaling might be the pathogenesis mechanism underlying the dysfunction of GLMN protein.

scholarly journals Targeted Next Generation Sequencing Identifies Novel Genetic Mutations in Patients with Waldenstrom's Macroglobulinemia/Lymphoplasmacytic Lymphoma or IgM-Monoclonal Gammopathies of Undetermined Significance

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2928-2928 ◽  
Author(s):  
Marzia Varettoni ◽  
Silvia Zibellini ◽  
Ettore Rizzo ◽  
Luca Malcovati ◽  
Virginia Valeria Ferretti ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Somatic Mutations ◽  
Genetic Mutations ◽  
Wild Type ◽  
Targeted Next Generation Sequencing ◽  
Coefficient Of Concordance ◽  
Generation Sequencing ◽  
Myd88 L265p ◽  
Undetermined Significance

Abstract Background. TheMYD88 (L265P) somatic mutation is present in more than 90% of patients (pts) with Waldenström's Macroglobulinemia (WM)/lymphoplasmacitic lymphoma (LPL). The second most common mutations are nonsense (NS) or frameshift (FS) mutations in the CXCR4 gene, detectable in approximately 25-30% of WM pts by Sanger sequencing. Limited data are available about other genetic mutations in WM/LPL and its precursor condition IgM-monoclonal gammopathy of undetermined significance (IgM-MGUS). Pts and methods. Using targeted next generation sequencing (NGS), we evaluated the prevalence of somatic mutations of 11 genes selected on the basis of evidences available from the literature (MYD88, CXCR4, ARID1A, KMT2D, TP53, NOTCH2, PRDM1, CD79b, TRAF3,TNFAIP3, MYDBBP1A) in 119 pts, classified as WM/LPL (n=63) or IgM-MGUS (n=56) according to International Consensus Criteria. Median age of pts (67 males, 52 females) was 65 years (range: 38-82). Samples were collected at diagnosis (n=101), after diagnosis but before any treatment (n=9) or at progression after therapy (n=9). Paired tumor and germline DNA extracted respectively from CD19-selected and CD19-depleted bone marrow (BM) mononuclear cells was available in all pts. Mean resequencing depth across gene panel was 1009x. Only mutations tagged as oncogenic or possibly oncogenic based on information derived from the literature and on in silico prediction effect were considered in the analysis. For MYD88 (L265P) and CXCR4 mutations, results obtained with NGS were compared with those obtained respectively with allele-specific PCR (AS-PCR) and Sanger sequencing. Results. Overall, we found 151 mutations in 88 pts (74%). The median number of mutations was significantly higher in WM/LPL as compared with IgM-MGUS and in pts previously treated as compared with untreated ones (median 2 versus 1, P < 0.001 for both comparisons). MYD88 mutations were found in 80/119 pts (67%), with a median allele burden of 34.2% (range: 2.5-93.3%). The prevalence of MYD88 mutations was significantly higher in WM/LPL as compared with IgM-MGUS (86% versus 46%, P <0.001). MYD88 mutations other than classical L265P (n=76) were found in 4 pts and were represented by V217F (n=2), S219C (n=1), M232T (n=1). Fifteen pts who were MYD88 (L265P) wild-type by NGS were found to be mutated by AS-PCR (K coefficient of concordance between NGS and AS-PCR: 70%, P < 0.001). CXCR4 mutations were found in 19/119 pts (16%), with a median allele burden of 34% (range: 4.2-84%). The prevalence of CXCR4 mutations was significantly higher in WM/LPL as compared with IgM-MGUS (24% versus 7%, P < 0.02). The K coefficient of concordance between NGS and Sanger was 83% (P < 0.001), with 2 pts mutated only by NGS and 2 pts mutated only by Sanger. Somatic mutations were also found in KMT2D (formerly known as MLL2) (16% of pts), TP53 (8%), NOTCH2 (7%), PRDM1 (4%), ARID1A (3%), CD79b (2%), and TRAF3 (1%). No mutations were found in MYBBP1A and TNFAI3. Overall, the prevalence of these mutations was significantly lower in pts wild-type either for MYD88 or CXCR4 as compared with those with MYD88 and/or CXCR4 mutations (15% versus 41%, P = 0.04). The prevalence of KMT2D mutations was significantly higher in WM/LPL as compared with MGUS (25% versus 5%), while for the other genes the distribution was not statistically different according to diagnosis. With a median follow-up of 20 months (range: 0-264), we did not find a statistically significant correlation between genetic mutations and pts' outcome in terms of overall survival or time to first treatment. Conclusions. In this cohort of pts with WM/LPL and IgM-MGUS studied with NGS we could demonstrate that: i) NGS identifies MYD88 mutations other than L265P in a small proportion of pts; ii) the prevalence of CXCR4 mutations by Sanger is confirmed by NGS, despite the higher sensitivity of the latter method; iii) the subgroup of pts wild type either for MYD88 or CXCR4 shows a low incidence of other genetic mutations; iv) 25% of pts with WM/LPL were found to carry KMT2D mutations, a prevalence similar to that reported in marginal zone lymphoma; v) genetic mutations are more common in WM/LPL than in IgM-MGUS in agreement with the hypothesis that multiple genetic hits are required for progression from a pre-benign condition to a neoplastic disease; vi) due to the indolent nature of these disorders, longer follow-up is probably needed to see the prognostic impact of these mutations, if any. Disclosures No relevant conflicts of interest to declare.

Exome Sequencing Identifies Recurring DNMT3A Mutations in Adult ETP-ALL

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1377-1377
Author(s):  
Martin Neumann ◽  
Sandra Heesch ◽  
Cornelia Schlee ◽  
Stefan Schwartz ◽  
Nicola Goekbuget ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Sanger Sequencing ◽  
Somatic Mutations ◽  
Pediatric Patients ◽  
Genetic Alterations ◽  
Whole Exome ◽  
Repressor Complex ◽  
Flt3 Mutations ◽  
Exome Sequence

Abstract Abstract 1377 Introduction: Early T-cell precursor (ETP) ALL accounting for 10% of all T-ALL cases is of special interest because of its proposed origin from early thymic progenitors with multilineage differentiation potential. ETP-ALL is associated with a poorer outcome in pediatric and adult patients. On the molecular level, ETP-ALL is characterized by a specific immunophenotype (CD1-, CD5weak, CD8-, co-expression of stem cell and/or myeloid antigens) and distinct molecular features (expression of stem cell genes, high frequency of FLT3 mutations with absence of NOTCH1 mutations). Whereas a highly heterogeneous genetic pattern was revealed by whole genome sequencing in pediatric patients, the genetic background of adult ETP-ALL remains largely unknown. Here we investigated genetic alterations in adult ETP-ALL by whole exome sequencing and subsequently analyzed specific target genes. Patients and methods: We performed whole exome sequencing of five paired (diagnosis/remission) adult ETP-ALL patients enrolled in German Acute Lymphoblastic Leukemia Multicenter Study Group (GMALL) trials. Using exon capturing from genomic DNA, followed by 76-bp paired-end sequencing on an Illumina Genome Analyzer IIx platform, we generated at least 5 Gb of exome sequence from each ETP-ALL and remission samples. Somatic mutations were identified by comparing the ETP-ALL with the remission exome sequence, excluding all annotated polymorphisms (dbSNP130), non-coding positions and positions with evidence of a variant in the corresponding remission samples. Candidate variants were confirmed by capillary sequencing of genomic DNA. The DNMT3A mutations status was analyzed by Sanger sequencing of exons 11–23 in additional 68 adult ETP-ALL (55 male, 13 female, median age: 38 years) as well as the mutation status of the polycomb repressor complex (PRC) genes EZH2 and SUZ12. For 52 of 68 patients clinical follow-up data were available. Results: Using whole exome sequencing we found a total of 56 non-synonymous somatic mutations or indels in the five ETP-ALL patients (range: 6 to 16 per patient). Eleven mutations/indels affected cancer genes. DNMT3A (2/5) and FAT3 (2/5) were recurrently mutated in the five patients. The DNA-methyl-transferase DNMT3A is a frequent mutational target in acute myeloid leukemia (AML; 20%), whereas FAT3 (FAT, tumor suppressor homolog 3) mutations were recently reported in ovarian carcinoma (TCGA, Nature 2011). Novel mutations identified in adult ETP-ALL involved genes in epigenetic regulation (e.g. MLL2, MLL3, BMI1), and in genes previously reported to be mutated in ETP-ALL (e.g. in JAK1, ETV6, NOTCH1, DNM2). By Sanger sequencing, we screened for DNMT3A mutations in a larger cohort of adult ETP-ALL. DNMT3A mutations were present in 11 of the 68 (16%) patients, a mutation rate similar to AML. Amino acid R882 (exon 23), the most frequently mutated amino acid in AML, was mutated in five ETP-ALL. The remaining six mutations occurred in single spots, with one exception in the ZNF or the MTF domain. Patients with a DNMT3A mutation were significantly older (median: 63 vs 37 years, P=0.016). No correlation was found between DNMT3A and FLT3 mutations (27% in DNMT3A mut pts. vs. 37% in DNMT3A wt pts., P=0.41) or NOTCH1 mutations (10% in DNMT3A mut pts. vs. 16% in DNMT3A wt pts., P=0.47). In addition, we investigated genetic alterations in epigenetic regulators including members of the polycomb repressor complex (PRC). Mutations were seen in EZH2 in 4/68 (6%), SUZ12 in 1/68 (1%) and SH2B3 in 4/69 (6%) of ETP-ALL. Interestingly, patients with at least one mutation in an epigenetic regulator gene (DNMT3A, SUZ12, SH2B3, MLL2, or EZH2) showed a trend towards an inferior survival (one-year-survival: 50% vs. 85%, P=0.08). Conclusion: Adult ETP-ALL patients display a heterogenous spectrum of mutations, particularly affecting genes involved in epigenetic regulation. The spectrum is different to pediatric patients with a lower rate of polycomb repressor complex and a higher rate of DNMT3A mutations. The higher rate of DNMT3A mutations in older patients might point to a different pathogenesis compared to pediatric ETP-ALL. Like in AML, DNMT3A mutations in adult ETP-ALL show a similar frequency, within the same hot spots and are correlated with an adverse prognostic value, underscoring the myeloid character of ETP-ALL. Thus, these data may provide a rationale to use epigenetic therapy in ETP-ALL. Disclosures: Krebs: Illumina: Honoraria. Greif:Illumina: Honoraria.

Somatic Activating Mutations In CXCR4 Are Common In Patients With Waldenstrom’s Macroglobulinemia, and Their Expression In WM Cells Promotes Resistance To Ibrutinib

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4424-4424 ◽  
Author(s):  
Yang Cao ◽  
Zachary Hunter ◽  
Xia Liu ◽  
Lian Xu ◽  
Guang Yang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Somatic Mutation ◽  
Sanger Sequencing ◽  
Somatic Mutations ◽  
Akt Signaling ◽  
Wild Type ◽  
Control Vector ◽  
Whim Syndrome ◽  
Frame Shift

Background Waldenstrom's macroglobulinemia (WM) is an indolent non-Hodgkin's lymphoma characterized by the accumulation of IgM secreting lymphoplasmacytic cells in the bone marrow. CXCR4 is a chemokine receptor that promotes the survival, migration, and adhesion to the bone marrow stroma of WM lymphoplasmacytic cells (LPC) through interactions with its ligand CXCL12. Through whole genome sequencing, we identified somatic mutations in CXCR4 that affected 1/3 of WM patients. These mutations were identical or functionally similar to those associated with Warts, Hypogammaglobulinemia, Infection, and Myelokathexis (WHIM) syndrome (Hunter et al, ASCO 2012), a rare autosomal dominant genetic disorder that is caused by frame shift or nonsense mutations in the carboxyl-terminal cytoplasmic tail of CXCR4. In WHIM syndrome, loss of the c-terminal tail of CXCR4 impairs receptor internalization, thereby prolonging G-protein and β-arrestin signaling (Lugane et al, Blood 2008). Ibrutinib induces WM cell death, and is highly active in WM (Treon et al, ICML-12, 2013). Since the target of ibrutinib (BTK) is a known downstream target of CXCR4, we sought to clarify if ibrutinib activity in WM LPCs was modulated by WHIM-like mutations in CXCR4. Methods We first sought to confirm the frequency of WHIM-like mutations in 87 untreated WM patients by Sanger sequencing. The most common CXCR4 somatic mutation identified (S338X) in these studies was then cloned by PCR from CD19+ LPCs from a WM patient with this somatic mutation. Wild type (WT) and S338X CXCR4 cDNAs were subcloned into plenti-IRES-GFP vector, and transduced using an optimized lentiviral based strategy into BCWM.1 WM cells. Five days after transduction, GFP positive cells were sorted and used for functional studies. Surface expression of CXCR4 was determined by flow cytometeric analysis using a PE-conjugated anti-CXCR4 monoclonal antibody. The expression of phosphorylated BTK, AKT, and ERK1/2 was determined by western blot analysis. Cell proliferation was measured with alamar blue. Results Sanger sequencing identified nonsense or frame shift mutations (WHIM-like) in the c-terminal tail of CXCR4 in 28 of 87 (32%) patients, the most common of which was a non-sense mutation (S338X) that was present in 12 patients. BCWM.1 cells were then transduced with control vector, CXCR4 wild type or CXCR4 S338X mutant expressing vectors. Expression was confirmed by cDNA Sanger sequencing. Stably transduced cells exposed to ibrutinib (0.5uM or 1uM) showed significantly reduced cell proliferation (p<0.005). Ibrutinib treated control vector and CXCR4 wild-type transduced cells showed suppressed tumor cell growth even in the presence of the CXCR4 ligand CXCL12 (20 nM), whereas cells transduced with CXCR4 S338X WHIM-like mutation demonstrated resistance to ibrutinib growth effect (p<0.005). In turn, this rescue could be blocked by treatment with 30uM of the CXCR4 specific inhibitor AMD3100 confirming that this effect was mediated through CXCR4 (p<0.005) (Figure 1). Phosphorylated BTK, ERK1/2 and AKT signaling increased following CXCL12 stimulation in all transduced cells, while ibrutinib inhibited their activation in control vector and CXCR4 wild-type, but not CXCR4 S338X mutant cells. CXCR4 triggered signaling by CXCL12 in these experiments was confirmed by pre-treatment with AMD3100. Conclusions By Sanger sequencing, WHIM-like CXCR4 somatic mutations are observed in 1/3 of untreated WM patients. WHIM-like CXCR4 mutations are associated with resistance to ibrutinib mediated ERK1/2 and AKT signaling, as well as growth suppression in the presence of the CXCR4 ligand, CXCL12, in WM cells. These studies may have important implications for CXCR4 modulation in the treatment of WM, as well as potential use of CXCR4 mutations in predicting outcome for patients undergoing ibrutinib therapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Coamplification at Lower Denaturation Temperature-PCR Increases Mutation-Detection Selectivity of TaqMan-Based Real-Time PCR

2009 ◽  
Vol 55 (4) ◽  
pp. 748-756 ◽  
Author(s):  
Jin Li ◽  
Lilin Wang ◽  
Pasi A Jänne ◽  
G Mike Makrigiorgos
Keyword(s):  
Somatic Mutations ◽  
Kinase Inhibitors ◽  
Hot Spot ◽  
Resistance Mutation ◽  
Clinical Samples ◽  
Nucleotide Polymorphisms ◽  
Wild Type ◽  
Denaturation Temperature ◽  
Low Level ◽  
Single Temperature

Abstract Background: DNA genotyping with mutation-specific TaqMan® probes (Applied Biosystems) is broadly used in detection of single-nucleotide polymorphisms but is less so for somatic mutations because of its limited selectivity for low-level mutations. We recently described coamplification at lower denaturation temperature-PCR (COLD-PCR), a method that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences during the PCR. We demonstrate that combining COLD-PCR with TaqMan technology provides TaqMan genotyping with the selectivity needed to detect low-level somatic mutations. Methods: Minor-groove binder-based or common TaqMan probes were designed to contain a nucleotide that matches the desired mutation approximately in the middle of the probe. The critical denaturation temperature (Tc) of each amplicon was then experimentally determined. COLD-PCR/TaqMan genotyping was performed in 2 steps: denaturation at the Tc, followed by annealing and extension at a single temperature (fast COLD-PCR). The threshold cycle was used to identify mutations on the basis of serial dilutions of mutant DNA into wild-type DNA and to identify TP53 (tumor protein p53) and EGFR [epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)] mutations in tumors. Results: COLD-PCR/TaqMan genotyping identified G&gt;A mutations within TP53 exon 8 (codon 273 mutation hot spot) and C&gt;T mutations within the EGFR gene (drug-resistance mutation T790M) with a selectivity improvement of 15- to 30-fold over regular PCR/TaqMan genotyping. A second round of COLD-PCR/TaqMan genotyping improved the selectivity by another 15- to 30-fold and enabled detection of 1 mutant in 2000 wild-type alleles. Use of COLD-PCR/TaqMan genotyping allowed quantitative identification of low-level TP53 and T790 mutations in colon tumor samples and in non-small-cell lung cancer cell lines treated with kinase inhibitors. Conclusions: The major improvement in selectivity provided by COLD-PCR enables the popular TaqMan genotyping method to become a powerful tool for detecting low-level mutations in clinical samples.

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