scholarly journals Detecting and Quantitating Low Fraction DNA Variants with Low-Depth Sequencing

2020 ◽  
Author(s):  
Ping Song ◽  
Sherry X. Chen ◽  
Yan Helen Yan ◽  
Alessandro Pinto ◽  
Lauren Y. Cheng ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
High Sensitivity ◽  
Human Cell Line ◽  
Clinical Samples ◽  
Single Nucleotide Variants ◽  
Tumor Biopsy ◽  
Single Nucleotide ◽  
Tissue Samples ◽  
Acquired Drug Resistance ◽  
Dna Variants

DNA sequence variants with low allele frequencies below 1% are difficult to detect and quantitate by sequencing, due to the intrinsic error of sequencing-by-synthesis (NGS). Unique molecular identifier barcodes can in principle help NGS detect mutations down to 0.1% variant allele frequency (VAF), but require extremely high sequencing depths of over 25,000x, rendering high sensitivity mutation detection out of reach for most research and clinical samples. Here, we present the multiplex blocker displacement amplification (mBDA) method to selectively enrich DNA variants by an average of 300-fold in highly multiplexed NGS settings. On a 80-plex human single nucleotide polymorphism panel, mBDA achieves a 0.019% VAF limit of detection for single nucleotide variants, using only 250x sequencing depth, and detects human cell line contamination down to 0.07%. Using this technology, we constructed a 16-plex melanoma NGS panel covering 145 actionable mutations across 9 genes, and applied it to 19 fresh/frozen tumor biopsy tissue samples with high tumor fractions. We found low VAF mutations (0.2% to 5%) in 37% of the samples (7/19, 95% confidence interval 19%-58%). These results suggest that tumor heterogeneity could be significantly more pervasive than previously recognized, and can contribute significantly to acquired drug resistance to targeted therapies. We also validate mBDA panels on clinical cell-free DNA samples from lung cancer patients.

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 SARS-CoV-2 Direct Detection Without RNA Isolation With Loop-Mediated Isothermal Amplification (LAMP) and CRISPR-Cas12

2021 ◽  
Vol 8 ◽  
Author(s):  
Alfredo Garcia-Venzor ◽  
Bertha Rueda-Zarazua ◽  
Eduardo Marquez-Garcia ◽  
Vilma Maldonado ◽  
Angelica Moncada-Morales ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Direct Detection ◽  
Direct Method ◽  
Rna Extraction ◽  
Rna Isolation ◽  
High Sensitivity ◽  
High Specificity ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Loop Mediated Isothermal Amplification

As to date, more than 49 million confirmed cases of Coronavirus Disease 19 (COVID-19) have been reported worldwide. Current diagnostic protocols use qRT-PCR for viral RNA detection, which is expensive and requires sophisticated equipment, trained personnel and previous RNA extraction. For this reason, we need a faster, direct and more versatile detection method for better epidemiological management of the COVID-19 outbreak. In this work, we propose a direct method without RNA extraction, based on the Loop-mediated isothermal amplification (LAMP) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein (CRISPR-Cas12) technique that allows the fast detection of SARS-CoV-2 from patient samples with high sensitivity and specificity. We obtained a limit of detection of 16 copies/μL with high specificity and at an affordable cost. The diagnostic test readout can be done with a real-time PCR thermocycler or with the naked eye in a blue-light transilluminator. Our method has been evaluated on a small set of clinical samples with promising results.

scholarly journals Epigenetic Markers for Molecular Detection of Prostate Cancer

2007 ◽  
Vol 23 (1-2) ◽  
pp. 31-41 ◽  
Author(s):  
Vera L. Costa ◽  
Rui Henrique ◽  
Carmen Jerónimo
Keyword(s):  
Prostate Cancer ◽  
Sensitivity And Specificity ◽  
Prostate Cancer Risk ◽  
High Sensitivity ◽  
Promoter Hypermethylation ◽  
Clinical Samples ◽  
Screening Methods ◽  
Tissue Samples ◽  
Age Related ◽  
Small Set

Prostate cancer is a highly prevalent malignancy, which is clinically silent but curable while organ-confined. Because available screening methods show poor sensitivity and specificity, the development of new molecular markers is warranted. Epigenetic alterations, mainly promoter hypermethylation of cancer-related genes, are common events in prostate cancer and might be used as cancer biomarkers. Moreover, the development of quantitative, high-throughput techniques to assess promoter methylation enabled the simultaneous screening of multiple clinical samples. From the numerous cancer-related genes hypermethylated in prostate cancer only a few proved to be strong candidates to become routine biomarkers. This small set of genes includesGSTP1,APC,RARβ2,Cyclin D2,MDR1, andPTGS2. Single and/or multigene analyses demonstrated the feasibility of detecting early prostate cancer, with high sensitivity and specificity, in body fluids (serum, plasma, urine, and ejaculates) and tissue samples. In addition, quantitative hypermethylation of several genes has been associated with clinicopathologic features of tumor aggressiveness, and also reported as independent prognostic factor for relapse. The identification of age-related methylation at specific loci and the differential frequency of methylation among ethnical groups, also provided interesting data linking methylation and prostate cancer risk. Although large trials are needed to validate these findings, the clinical use of these markers might be envisaged for the near future.

scholarly journals High Sensitivity Sanger Sequencing Detection of BRAF Mutations in Metastatic Melanoma FFPE Tissue Specimens

2020 ◽  
Author(s):  
Lauren Cheng ◽  
Lauren Haydu ◽  
Ping Song ◽  
Jianyi Nie ◽  
Michael Tetzlaff ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Limit Of Detection ◽  
False Negative ◽  
High Sensitivity ◽  
Ffpe Tissue ◽  
Braf Mutations ◽  
Tissue Samples ◽  
Ffpe Samples ◽  
Droplet Pcr ◽  
Tissue Specimens

Abstract Mutations 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 VarTraceTM BRAF assay, based on the Blocker Displacement Amplification (BDA) technology, is capable of detecting BRAF V600 mutations down to 0.26% 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 Validation of an NGS Panel Designed for Detection of Actionable Mutations in Tumors Common in Latin America

2021 ◽  
Vol 11 (9) ◽  
pp. 899
Author(s):  
Mauricio Salvo ◽  
Evelin González-Feliú ◽  
Jessica Toro ◽  
Iván Gallegos ◽  
Ignacio Maureira ◽  
...  
Keyword(s):  
Latin America ◽  
Latin American ◽  
Hedgehog Signaling ◽  
Predictive Biomarkers ◽  
High Sensitivity ◽  
Clinical Samples ◽  
Dna Integrity ◽  
Precision Oncology ◽  
Single Nucleotide Variants ◽  
Sensitivity Specificity

Next-generation sequencing (NGS) is progressively being used in clinical practice. However, several barriers preclude using this technology for precision oncology in most Latin American countries. To overcome some of these barriers, we have designed a 25-gene panel that contains predictive biomarkers for most current and near-future available therapies in Chile and Latin America. Library preparation was optimized to account for low DNA integrity observed in formalin-fixed paraffin-embedded tissue. The workflow includes an automated bioinformatic pipeline that accounts for the underrepresentation of Latin Americans in genome databases. The panel detected small insertions, deletions, and single nucleotide variants down to allelic frequencies of 0.05 with high sensitivity, specificity, and reproducibility. The workflow was validated in 272 clinical samples from several solid tumor types, including gallbladder (GBC). More than 50 biomarkers were detected in these samples, mainly in BRCA1/2, KRAS, and PIK3CA genes. In GBC, biomarkers for PARP, EGFR, PIK3CA, mTOR, and Hedgehog signaling inhibitors were found. Thus, this small NGS panel is an accurate and sensitive method that may constitute a more cost-efficient alternative to multiple non-NGS assays and costly, large NGS panels. This kind of streamlined assay with automated bioinformatics analysis may facilitate the implementation of precision medicine in Latin America.

scholarly journals Exome sequencing of glioblastoma-derived cancer stem cells reveals rare clinically relevant frameshift deletion in MLLT1 gene

2022 ◽  
Vol 22 (1) ◽  
Author(s):  
Hany E. Marei ◽  
Asmaa Althani ◽  
Nahla Afifi ◽  
Anwarul Hasan ◽  
Thomas Caceci ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cancer Stem Cells ◽  
Allelic Frequency ◽  
Response To Therapy ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Tissue Samples ◽  
Frameshift Deletion ◽  
Prognostic Outcome ◽  
The Common

Abstract Background Glioblastoma multiforme (GBM) is a heterogeneous CNS neoplasm which causes significant morbidity and mortality. One reason for the poor prognostic outcome of GBM is attributed to the presence of cancer stem cells (CSC) which confer resistance against standard chemo- and radiotherapeutics modalities. Two types of GBM-associated CSC were isolated from the same patient: tumor core- (c-CSC) and peritumor tissue-derived cancer stem cells (p-CSC). Our experiments are focused on glioblastoma–IDH-wild type, and no disease-defining alterations were present in histone, BRAF or other genes. Methods In the present study, potential differences in genetic variants between c-CSC versus p-CSC derived from four GBM patients were investigated with the aims of (1) comparing the exome sequences between all the c-CSC or p-CSC to identify the common variants; (2) identifying the variants affecting the function of genes known to be involved in cancer origin and development. Results By comparative analyses, we identified common gene single nucleotide variants (SNV) in all GBM c-CSC and p-CSC, a potentially deleterious variant was a frameshift deletion at Gln461fs in the MLLT1 gene, that was encountered only in p-CSC samples with different allelic frequency. Conclusions We discovered a potentially harmful frameshift deletion at Gln461fs in the MLLT1 gene. Further investigation is required to confirm the presence of the identified mutations in patient tissue samples, as well as the significance of the frameshift mutation in the MLLT1 gene on GBM biology and response to therapy based on genomic functional experiments.

scholarly journals Diagnostic performance of rapid antigen tests (RATs) for SARS-CoV-2 and their efficacy in monitoring the infectiousness of COVID-19 patients

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
John G. Routsias ◽  
Maria Mavrouli ◽  
Panagiota Tsoplou ◽  
Kyriaki Dioikitopoulou ◽  
Athanasios Tsakris
Keyword(s):  
Diagnostic Performance ◽  
Limit Of Detection ◽  
High Sensitivity ◽  
Cost Effective ◽  
Clinical Samples ◽  
True Positive ◽  
Antigen Tests ◽  
The Cost ◽  
Very High ◽  
Test Result

AbstractThe most widely used test for the diagnosis of SARS-CoV-2 infection is a PCR test. PCR has very high sensitivity and is able to detect very low amounts of RNA. However, many individuals receiving a positive test result in a context of a PCR-based surveillance might be infected with SARS-CoV-2, but they are not contagious at the time of the test. The question arises regards if the cost effective, portable rapid antigen tests (RATs) have a better performance than PCR in identification of infectious individuals. In this direction, we examined the diagnostic performance of RATs from 14 different manufacturers in 400 clinical samples with known rRT-PCR cycles threshold (cT) and 50 control samples. Substantial variability was observed in the limit of detection (LOD) of different RATs (cT = 26.8–34.7). The fluorescence-based RAT exhibited a LOD of cT = 34.7. The use of the most effective RATs leads to true positive rates (sensitivities) of 99.1% and 90.9% for samples with cT ≤ 30 and cT ≤ 33, respectively, percentages that can guarantee a sensitivity high enough to identify contagious patients. RAT testing may also substantially reduce the quarantine period for infected individuals without compromising personal or public safety.

Technical validation of a high-sensitivity target capture NGS assay using unique molecular identifier approach.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e13657-e13657
Author(s):  
Ruifang Mao ◽  
Shanshan Xiao ◽  
Rui Lin ◽  
Yuchen Wang ◽  
Tao Wang
Keyword(s):  
Copy Number ◽  
Somatic Mutations ◽  
Limit Of Detection ◽  
Sequence Data ◽  
Nsclc Patient ◽  
High Sensitivity ◽  
Alternative Methods ◽  
Driver Mutations ◽  
Single Nucleotide Variants ◽  
Illumina Hiseq

e13657 Background: Identification of a broad spectrum of somatic mutations is crucial to guide targeted therapy such as for non-small cell lung cancer (NSCLC) patients. In the clinical environment, it requires well validated NGS workflow both for the wet-lab and dry-lab procedures. Here we describe a high sensitivity target NGS assay to accurately capture single nucleotide variants (SNVs), short insertions and deletions (indels), copy number alterations and gene rearrangements for formalin-fixed paraffin-embedded (FFPE) NSCLC patient samples. Extensive analytical validation was performed following the checklists of College of American Pathologists. Methods: Next generation sequencing (NGS) libraries were prepared using extracted DNA from FFPE tissue NSCLC patient samples. The protocol for library generation was optimized in several steps and incorporated 10bp unique molecular identifiers (UMIs). The libraries were sequenced on Illumina HiSeq X-Ten platform. The sequence data was analyzed by an in-house bioinformatics pipeline to call somatic mutations at an average depth of 4000X. Results: We tested the accuracy of 68 clinical tumor samples that were also validated by conventional or alternative methods in the third party CAP accredited labs. We observed 100% sensitivity and 100% specificity compared with the other lab¡¯s validation results. To define the limit of detection (LOD) for different mutation types, clinical DNA samples containing different variants were diluted with normal DNA. The LODs for SNV (as in EGFR L858R) and indel (as in EGFR 19del) were 0.5% and 1%, respectively. Addressing the LOD of fusion and copy number alteration is usually challenging. Our NGS assay was able to achieve 2% LOD for gene rearrangement (fusion) and 3.5 copies for copy number amplification. The high reproducibility was also achieved by inter- and intra- replicate experiments. Our NGS assay showed better performance than other widely used commercial NGS assay panels. Conclusions: We have validated an NGS based approach with UMI technology that is able to achieve high accuracy and sensitivity as low as 0.5% for detection of somatic mutations, which will improve the clinical testing performance for NSCLC FFPE samples with low allele frequencies of driver mutations.

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