scholarly journals Confirming putative variants at ≤ 5% allele frequency using allele enrichment and Sanger sequencing

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yan Helen Yan ◽  
Sherry X. Chen ◽  
Lauren Y. Cheng ◽  
Alyssa Y. Rodriguez ◽  
Rui Tang ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Limit Of Detection ◽  
High Sensitivity ◽  
Sequencing Errors ◽  
Whole Exome ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Fresh Frozen ◽  
The Cost ◽  
Allelic Fraction

AbstractWhole exome sequencing (WES) is used to identify mutations in a patient’s tumor DNA that are predictive of tumor behavior, including the likelihood of response or resistance to cancer therapy. WES has a mutation limit of detection (LoD) at variant allele frequencies (VAF) of 5%. Putative mutations called at ≤ 5% VAF are frequently due to sequencing errors, therefore reporting these subclonal mutations incurs risk of significant false positives. Here we performed ~ 1000 × WES on fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissue biopsy samples from a non-small cell lung cancer patient, and identified 226 putative mutations at between 0.5 and 5% VAF. Each variant was then tested using NuProbe NGSure, to confirm the original WES calls. NGSure utilizes Blocker Displacement Amplification to first enrich the allelic fraction of the mutation and then uses Sanger sequencing to determine mutation identity. Results showed that 52% of the 226 (117) putative variants were disconfirmed, among which 2% (5) putative variants were found to be misidentified in WES. In the 66 cancer-related variants, the disconfirmed rate was 82% (54/66). This data demonstrates Blocker Displacement Amplification allelic enrichment coupled with Sanger sequencing can be used to confirm putative mutations ≤ 5% VAF. By implementing this method, next-generation sequencing can reliably report low-level variants at a high sensitivity, without the cost of high sequencing depth.

Confirming Putative Variants at ≤5% Allele Frequency Using Allele Enrichment and Sanger Sequencing

2021 ◽  
Author(s):  
Yan Helen Yan ◽  
Sherry X. Chen ◽  
Lauren Y. Cheng ◽  
Alyssa Y. Rodriguez ◽  
Rui Tang ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Limit Of Detection ◽  
High Sensitivity ◽  
Sequencing Errors ◽  
Whole Exome ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Fresh Frozen ◽  
The Cost ◽  
Allelic Fraction

Abstract Whole exome sequencing (WES) is used to identify mutations in a patient’s tumor DNA that are predictive of tumor behavior, including the likelihood of response or resistance to cancer therapy. WES has a mutation limit of detection (LoD) at variant allele frequencies (VAF) of 5%. Putative mutations called at ≤5% VAF are frequently due to sequencing errors, therefore reporting these subclonal mutations incurs risk of significant false positives. Here we performed ~1000x WES on fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissue biopsy samples from a non-small cell lung cancer patient, and identified 226 putative mutations at between 0.5% and 5% VAF. Each variant was then tested using NuProbe NGSureTM, to confirm the original WES calls. NGSure utilizes Blocker Displacement Amplification to first enrich the allelic fraction of the mutation and then uses Sanger sequencing to determine mutation identity. Results showed that 52% of the 226 (117) putative variants were disconfirmed, among which 2% (5) putative variants were found to be misidentified in WES. In the 66 cancer-related variants, the disconfirmed rate was 82% (54/66). This data demonstrates Blocker Displacement Amplification allelic enrichment coupled with Sanger sequencing can be used to confirm putative mutations ≤5% VAF. By implementing this method, next-generation sequencing can reliably report low-level variants at a high sensitivity, without the cost of high sequencing depth.

scholarly journals Comparison of Accuracy of Whole-Exome Sequencing with Formalin-Fixed Paraffin-Embedded and Fresh Frozen Tissue Samples

PLoS ONE ◽  
2015 ◽  
Vol 10 (12) ◽  
pp. e0144162 ◽  
Author(s):  
Ensel Oh ◽  
Yoon-La Choi ◽  
Mi Jeong Kwon ◽  
Ryong Nam Kim ◽  
Yu Jin Kim ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Tissue Samples ◽  
Whole Exome ◽  
Formalin Fixed Paraffin ◽  
Frozen Tissue ◽  
Formalin Fixed Paraffin Embedded ◽  
Fresh Frozen ◽  
Formalin Fixed

scholarly journals Assessment of a High Sensitivity Method for Identification of IDH1 R132x Mutations in Tumors and Plasma of Intrahepatic Cholangiocarcinoma Patients

Cancers ◽  
2019 ◽  
Vol 11 (4) ◽  
pp. 454 ◽  
Author(s):  
Caterina Peraldo-Neia ◽  
Maria Scatolini ◽  
Enrico Grosso ◽  
Pasquale Lombardi ◽  
Roberto Filippi ◽  
...  
Keyword(s):  
Disease Progression ◽  
Intrahepatic Cholangiocarcinoma ◽  
Nested Pcr ◽  
Sanger Sequencing ◽  
High Efficiency ◽  
High Sensitivity ◽  
Circulating Free Dna ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded

Hotspot codon 132 mutations (R132xIDH1m) are frequent in intrahepatic cholangiocarcinoma (ICC), are druggable by anti-IDH1m agents, and could represent a marker of disease progression. Developing an assay to identify R132xIDH1m would provide a useful tool to select patients benefitting from targeted treatments. We tested a quantitative real-time allele-specific polymerase chain reaction (qPCR)-based method to detect the main R132xIDH1m in an Italian ICC series (n = 61) of formalin-fixed paraffin-embedded (FFPE) samples, and on circulating-free DNA samples. The outcomes were compared with nested PCR/Sanger sequencing. Reconstitution experiments of plasmids harboring the different R132xIDH1m mixed with wild-type (WT) DNA demonstrated that qPCR is able to detect at least 2% of all mutated allele. High efficiency was also observed on patient-derived mutated DNA mixed with WT DNA (up to 10% and 0.3 ng of mutated template); qPCR detected 16.4% of mutated samples (one R132G, three R132C and six R132L) while nested PCR/Sanger sequencing only 8.2% (four R132L and one R132G). In a single patient with an R132C-mutated tumor, qPCR was also performed on plasma samples collected at four time-points, observing an increase correlating with disease progression. In conclusion, we developed a qPCR assay which could represent a fast, inexpensive and sensitive tool both for detection of R132xIDH1m in ICC samples and monitoring disease progression from liquid biopsy.

scholarly journals MicroSEC: Sequence error filtering pipeline for formalin-fixed and paraffin-embedded samples

2021 ◽  
Author(s):  
Masachika Ikegami ◽  
Shinji Kohsaka ◽  
Takeshi Hirose ◽  
Toshihide Ueno ◽  
Naoki Kanomata ◽  
...  
Keyword(s):  
Uneven Distribution ◽  
Clinical Sequencing ◽  
Sequencing Errors ◽  
Sequence Error ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Fresh Frozen ◽  
Formalin Fixed ◽  
Error Filtering

Abstract The clinical sequencing of tumors is usually performed on formalin-fixed, paraffin-embedded (FFPE) samples and results in many sequencing errors. Most of these errors are detected in chimeric reads caused by single-strand DNA molecules with microhomology. Our filtering pipeline, MicroSEC, focuses on the uneven distribution of mutations in each read and removes the sequencing errors in FFPE samples without eliminating the true mutations that are also detected in fresh frozen samples.

scholarly journals MicroSEC filters sequence errors for formalin-fixed and paraffin-embedded samples

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Masachika Ikegami ◽  
Shinji Kohsaka ◽  
Takeshi Hirose ◽  
Toshihide Ueno ◽  
Satoshi Inoue ◽  
...  
Keyword(s):  
Confidence Interval ◽  
Single Strand ◽  
Dna Molecules ◽  
Clinical Sequencing ◽  
Sequencing Errors ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Fresh Frozen ◽  
The Individual ◽  
Formalin Fixed

AbstractThe clinical sequencing of tumors is usually performed on formalin-fixed, paraffin-embedded samples and results in many sequencing errors. We identified that most of these errors are detected in chimeric reads caused by single-strand DNA molecules with microhomology. During the end-repair step of library preparation, mutations are introduced by the mis-annealing of two single-strand DNA molecules comprising homologous sequences. The mutated bases are distributed unevenly near the ends in the individual reads. Our filtering pipeline, MicroSEC, focuses on the uneven distribution of mutations in each read and removes the sequencing errors in formalin-fixed, paraffin-embedded samples without over-eliminating the mutations detected also in fresh frozen samples. Amplicon-based sequencing using 97 mutations confirmed that the sensitivity and specificity of MicroSEC were 97% (95% confidence interval: 82–100%) and 96% (95% confidence interval: 88–99%), respectively. Our pipeline will increase the reliability of the clinical sequencing and advance the cancer research using formalin-fixed, paraffin-embedded samples.

scholarly journals High sensitivity sanger sequencing detection of BRAF mutations in metastatic melanoma FFPE tissue specimens

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Lauren Y. Cheng ◽  
Lauren E. Haydu ◽  
Ping Song ◽  
Jianyi Nie ◽  
Michael T. Tetzlaff ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Limit Of Detection ◽  
False Negative ◽  
High Sensitivity ◽  
Ffpe Tissue ◽  
Braf Mutations ◽  
Tissue Samples ◽  
Ffpe Samples ◽  
Droplet Pcr ◽  
Tissue Specimens

AbstractMutations in the BRAF gene at or near the p. V600 locus are informative for therapy selection, but current methods for analyzing FFPE tissue DNA generally have a limit of detection of 5% variant allele frequency (VAF), or are limited to the single variant (V600E). These can result in false negatives for samples with low VAFs due to low tumor content or subclonal heterogeneity, or harbor non-V600 mutations. Here, we show that Sanger sequencing using the NuProbe VarTrace BRAF assay, based on the Blocker Displacement Amplification (BDA) technology, is capable of detecting BRAF V600 mutations down to 0.20% VAF from FFPE lymph node tissue samples. Comparison experiments on adjacent tissue sections using BDA Sanger, immunohistochemistry (IHC), digital droplet PCR (ddPCR), and NGS showed 100% concordance among all 4 methods for samples with BRAF mutations at ≥ 1% VAF, though ddPCR did not distinguish the V600K mutation from the V600E mutation. BDA Sanger, ddPCR, and NGS (with orthogonal confirmation) were also pairwise concordant for lower VAF mutations down to 0.26% VAF, but IHC produced a false negative. Thus, we have shown that Sanger sequencing can be effective for rapid detection and quantitation of multiple low VAF BRAF mutations from FFPE samples. BDA Sanger method also enabled detection and quantitation of less frequent, potentially actionable non-V600 mutations as demonstrated by synthetic samples.

scholarly journals Molecular Profiles and Metastasis Markers in Chinese Patients with Gastric Carcinoma

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Chao Chen ◽  
Chunmei Shi ◽  
Xiaochun Huang ◽  
Jianwei Zheng ◽  
Zhongyi Zhu ◽  
...  
Keyword(s):  
Gastric Carcinoma ◽  
Chinese Patients ◽  
Tissue Samples ◽  
Whole Exome ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
High Concordance ◽  
Predictive Role ◽  
Molecular Profiles ◽  
Genomic Regions

Abstract The goal of this work was to investigate the molecular profiles and metastasis markers in Chinese patients with gastric carcinoma (GC). In total, we performed whole exome sequencing (WES) on 74 GC patients with tumor and adjacent normal formalin-fixed, paraffin-embedded (FFPE) tissue samples. The mutation spectrum of these samples showed a high concordance with TCGA and other studies on GC. PTPRT is significantly associated with metastasis of GC, suggesting its predictive role in metastasis of GC. Patients carrying BRCA2 mutations tend not to metastasize, which may be related to their sensitivity to chemotherapy. Mutations in MACF1, CDC27, HMCN1, CDH1 and PDZD2 were moderately enriched in peritoneal metastasis (PM) samples. Furthermore, we found two genomic regions (1p36.21 and Xq26.3) were associated with PM of GC, and patients with amplification of 1p36.21 and Xq26.3 have a worse prognosis (P = 0.002, 0.01, respectively). Our analysis provides GC patients with potential markers for single and combination therapies.

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