scholarly journals Oncogene Concatenated Enriched Amplicon Nanopore Sequencing for Rapid, Accurate, and Affordable Somatic Mutation Detection

2020 ◽  
Author(s):  
Deepak Thirunavukarasu ◽  
Lauren Y. Cheng ◽  
Ping Song ◽  
Sherry X. Chen ◽  
Mitesh J. Borad ◽  
...  
Keyword(s):  
Somatic Mutations ◽  
Limit Of Detection ◽  
Digital Pcr ◽  
Nanopore Sequencing ◽  
Single Nucleotide ◽  
Tissue Samples ◽  
Recurrent Mutations ◽  
Ffpe Tumor ◽  
Somatic Mutation Detection ◽  
Intrinsic Error

Nanopore sequencing is more than 10-fold faster than sequencing-by-synthesis and provides reads that are roughly 100-fold longer. However, nanopore sequencing’s 7.5% intrinsic error rate renders it difficult to call somatic mutations with low variant allele frequencies (VAFs) without significant false positives. Here, we introduce the Oncogene Concatenated Enriched Amplicon Nanopore Sequencing (OCEANS) method, in which variants with low VAFs are selectively amplified and subsequently concatenated for nanopore sequencing. OCEANS allows accurate detection of somatic mutations with VAF limits of detection between 0.05% and ≤ 1%. We constructed 4 distinct multi-gene OCEANS panels targeting recurrent mutations in acute myeloid leukemia, melanoma, non-small-cell lung cancer, and hepatocellular carcinoma. Comparison experiments against Illumina NGS showed 99.79% to 99.99% area under the receiver-operator curve for these panels on clinical FFPE tumor samples. Furthermore, we identified a significant number of mutations below the standard NGS limit of detection in clinical tissue samples using each OCEANS panel. Comparison against digital PCR on 10 of putative mutations at ≤1% VAF showed 9 concordant positive calls with VAFs between 0.02% and 0.66%. By overcoming the primary challenge of nanopore sequencing on detecting low VAF single nucleotide variant mutations, OCEANS is poised to enable same-day clinical sequencing panels.

scholarly journals Oncogene Concatenated Enriched Amplicon Nanopore Sequencing for rapid, accurate, and affordable somatic mutation detection

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Deepak Thirunavukarasu ◽  
Lauren Y. Cheng ◽  
Ping Song ◽  
Sherry X. Chen ◽  
Mitesh J. Borad ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Somatic Mutations ◽  
Mutation Detection ◽  
Clinical Samples ◽  
Nanopore Sequencing ◽  
Small Cell Lung ◽  
Variant Allele Frequency ◽  
Single Nucleotide ◽  
Recurrent Mutations ◽  
Somatic Mutation Detection

AbstractWe develop the Oncogene Concatenated Enriched Amplicon Nanopore Sequencing (OCEANS) method, in which variants with low variant allele frequency (VAFs) are amplified and subsequently concatenated for Nanopore Sequencing. OCEANS allows accurate detection of somatic mutations with VAF limits of detection between 0.05 and 1%. We construct 4 distinct multi-gene OCEANS panels targeting recurrent mutations in acute myeloid leukemia, melanoma, non-small- cell lung cancer, and hepatocellular carcinoma and validate them on clinical samples. By demonstrating detection of low VAF single nucleotide variant mutations using Nanopore Sequencing, OCEANS is poised to enable same-day clinical sequencing panels.

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 Multiplex Droplet Digital PCR Quantification of Recurrent Somatic Mutations in Diffuse Large B-Cell and Follicular Lymphoma

2016 ◽  
Vol 62 (9) ◽  
pp. 1238-1247 ◽  
Author(s):  
Miguel Alcaide ◽  
Stephen Yu ◽  
Kevin Bushell ◽  
Daniel Fornika ◽  
Julie S Nielsen ◽  
...  
Keyword(s):  
Follicular Lymphoma ◽  
B Cell ◽  
Somatic Mutations ◽  
B Cell Lymphoma ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Analytical Sensitivity ◽  
Wild Type ◽  
Single Nucleotide ◽  
Large B Cell

Abstract BACKGROUND A plethora of options to detect mutations in tumor-derived DNA currently exist but each suffers limitations in analytical sensitivity, cost, or scalability. Droplet digital PCR (ddPCR) is an appealing technology for detecting the presence of specific mutations based on a priori knowledge and can be applied to tumor biopsies, including formalin-fixed paraffin embedded (FFPE) tissues. More recently, ddPCR has gained popularity in its utility in quantifying circulating tumor DNA. METHODS We have developed a suite of novel ddPCR assays for detecting recurrent mutations that are prevalent in common B-cell non-Hodgkin lymphomas (NHLs), including diffuse large B-cell lymphoma, follicular lymphoma, and lymphoplasmacytic lymphoma. These assays allowed the differentiation and counting of mutant and wild-type molecules using one single hydrolysis probe. We also implemented multiplexing that allowed the simultaneous detection of distinct mutations and an “inverted” ddPCR assay design, based on employing probes matching wild-type alleles, capable of detecting the presence of multiple single nucleotide polymorphisms. RESULTS The assays successfully detected and quantified somatic mutations commonly affecting enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) (Y641) and signal transducer and activator of transcription 6 (STAT6) (D419) hotspots in fresh tumor, FFPE, and liquid biopsies. The “inverted” ddPCR approach effectively reported any single nucleotide variant affecting either of these 2 hotspots as well. Finally, we could effectively multiplex hydrolysis probes targeting 2 additional lymphoma-related hotspots: myeloid differentiation primary response 88 (MYD88; L265P) and cyclin D3 (CCND3; I290R). CONCLUSIONS Our suite of ddPCR assays provides sufficient analytical sensitivity and specificity for either the invasive or noninvasive detection of multiple recurrent somatic mutations in B-cell NHLs.

Sensitive detection of tumor nucleic acids in plasma by mutation-enriched next generation sequencing.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e22041-e22041
Author(s):  
Andre Marziali
Keyword(s):  
Nucleic Acid ◽  
Dna Sequencing ◽  
Somatic Mutations ◽  
Mutation Detection ◽  
Limit Of Detection ◽  
Tumour Tissue ◽  
Sequencing Error ◽  
Next Generation ◽  
Tissue Samples ◽  
Electrophoretic Method

e22041 Background: Next Generation DNA Sequencing (NGS) is becoming the new standard for mutational profiling of tumour tissue, due to its flexibility, speed, and decreasing cost. While generally exceptional in performance, NGS suffers from a sequencing error rate of 0.1% – 1%, largely due to amplification-induced artifacts in its workflow. While this does not constitute a significant problem in application of NGS to sequencing of tumour tissue, it makes NGS impractical as a method to search for low abundance mutation signatures in plasma samples. Numerous publications have shown the presence of tumour signatures in the cell-free DNA (cfDNA) circulating in plasma, but concordance between the tumour signature and the plasma signature has been limited. This is likely due to limitations in the detection technologies used to search for cfDNA in plasma. To maximize concordance between plasma and tissue, it will be essential that sensitivities reaching 0.01% and below (as little as a single tumour mutant allele per sample) be achieved, and ideally that multiple mutational hot spots be analysed to maximize the chance of detection. Current technologies are incapable of such sensitivity over a large number of mutation loci. Methods: We have developed a novel electrophoretic method that can enrich nucleic acid samples over 1,000,000-fold for up to 100 somatic mutations, enabling reliable profiling of samples containing as little as 0.01% mutant. By enriching nucleic acid samples for specific targets prior to amplification and sequencing, we enable the use of NGS in plasma-based mutation detection and profiling. Results: We present technical and clinical data demonstrating highly sensitive multiplexed mutation detection in plasma and tissue samples, demonstrating 0.01% sensitivity over 45 somatic mutations per sample. Conclusions: We have demonstrated a novel somatic mutation enrichment methodology that allows DNA sequencing to work beyond its usual limit of detection to accurately profile solid tumours by detecting their mutation signature in plasma, even when the tumour DNA is present in plasma at abundances below 0.01%.

scholarly journals Donut PCR: a rapid, portable, multiplexed, and quantitative DNA detection platform with single-nucleotide specificity

2020 ◽  
Author(s):  
Dmitriy Khodakov ◽  
Jiaming Li ◽  
Jinny X. Zhang ◽  
David Yu Zhang
Keyword(s):  
Dynamic Range ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Turnaround Time ◽  
Isothermal Amplification ◽  
Nucleic Acid Detection ◽  
Nanopore Sequencing ◽  
Bacterial Identification ◽  
Single Nucleotide ◽  
Nucleotide Specificity

Current platforms for molecular analysis of DNA markers are either limited in multiplexing (qPCR, isothermal amplification), turnaround time (microarrays, NGS), quantitation accuracy (isothermal amplification, microarray, nanopore sequencing), or specificity against single-nucleotide differences (microarrays, nanopore sequencing). Here, we present the Donut PCR platform that features high multiplexing, rapid turnaround times, single nucleotide discrimination, and precise quantitation of DNA targets in a portable, affordable, and battery-powered instrument using closed consumables that minimize contamination. We built a bread-board instrument prototype and three assays/chips to demonstrate the capabilities of Donut PCR: (1) a 9-plex mammal identification panel, (2) a 15-plex bacterial identification panel, and (3) a 30-plex human SNP genotyping assay. The limit of detection of the platform is under 10 genomic copies in under 30 minutes, and the quantitative dynamic range is at least 4 logs. We envision that this platform would be useful for a variety of applications where rapid and highly multiplexed nucleic acid detection is needed at the point of care.

scholarly journals A verified genomic reference sample for assessing performance of cancer panels detecting small variants of low allele frequency

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Wendell Jones ◽  
Binsheng Gong ◽  
Natalia Novoradovskaya ◽  
Dan Li ◽  
Rebecca Kusko ◽  
...  
Keyword(s):  
Quality Control ◽  
Allele Frequency ◽  
Liquid Biopsy ◽  
Limit Of Detection ◽  
Reference Sample ◽  
Digital Pcr ◽  
Detection Sensitivity ◽  
Analytical Accuracy ◽  
And Performance ◽  
Reference Samples

Abstract Background Oncopanel genomic testing, which identifies important somatic variants, is increasingly common in medical practice and especially in clinical trials. Currently, there is a paucity of reliable genomic reference samples having a suitably large number of pre-identified variants for properly assessing oncopanel assay analytical quality and performance. The FDA-led Sequencing and Quality Control Phase 2 (SEQC2) consortium analyze ten diverse cancer cell lines individually and their pool, termed Sample A, to develop a reference sample with suitably large numbers of coding positions with known (variant) positives and negatives for properly evaluating oncopanel analytical performance. Results In reference Sample A, we identify more than 40,000 variants down to 1% allele frequency with more than 25,000 variants having less than 20% allele frequency with 1653 variants in COSMIC-related genes. This is 5–100× more than existing commercially available samples. We also identify an unprecedented number of negative positions in coding regions, allowing statistical rigor in assessing limit-of-detection, sensitivity, and precision. Over 300 loci are randomly selected and independently verified via droplet digital PCR with 100% concordance. Agilent normal reference Sample B can be admixed with Sample A to create new samples with a similar number of known variants at much lower allele frequency than what exists in Sample A natively, including known variants having allele frequency of 0.02%, a range suitable for assessing liquid biopsy panels. Conclusion These new reference samples and their admixtures provide superior capability for performing oncopanel quality control, analytical accuracy, and validation for small to large oncopanels and liquid biopsy assays.

scholarly journals Differential Expression of BARD1 Isoforms in Melanoma

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 320
Author(s):  
Lorissa I. McDougall ◽  
Ryan M. Powell ◽  
Magdalena Ratajska ◽  
Chi F. Lynch-Sutherland ◽  
Sultana Mehbuba Hossain ◽  
...  
Keyword(s):  
Long Range ◽  
Patient Outcomes ◽  
Splice Variants ◽  
Significant Proportion ◽  
Nanopore Sequencing ◽  
Rna Seq ◽  
Splice Isoforms ◽  
Tissue Samples ◽  
Multiple Transcripts ◽  
Mrna Variants

Melanoma comprises <5% of cutaneous malignancies, yet it causes a significant proportion of skin cancer-related deaths worldwide. While new therapies for melanoma have been developed, not all patients respond well. Thus, further research is required to better predict patient outcomes. Using long-range nanopore sequencing, RT-qPCR, and RNA sequencing analyses, we examined the transcription of BARD1 splice isoforms in melanoma cell lines and patient tissue samples. Seventy-six BARD1 mRNA variants were identified in total, with several previously characterised isoforms (γ, φ, δ, ε, and η) contributing to a large proportion of the expressed transcripts. In addition, we identified four novel splice events, namely, Δ(E3_E9), ▼(i8), IVS10+131▼46, and IVS10▼176, occurring in various combinations in multiple transcripts. We found that short-read RNA-Seq analyses were limited in their ability to predict isoforms containing multiple non-contiguous splicing events, as compared to long-range nanopore sequencing. These studies suggest that further investigations into the functional significance of the identified BARD1 splice variants in melanoma are warranted.

scholarly journals A simple method to estimate the in-house limit of detection for genetic mutations with low allele frequencies in whole-exome sequencing analysis by next-generation sequencing

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Takumi Miura ◽  
Satoshi Yasuda ◽  
Yoji Sato
Keyword(s):  
Next Generation Sequencing ◽  
Allele Frequency ◽  
Somatic Mutations ◽  
Limit Of Detection ◽  
Allele Frequencies ◽  
Genetic Mutations ◽  
Sequencing Data ◽  
Simple Method ◽  
Whole Exome ◽  
Generation Sequencing

Abstract Background Next-generation sequencing (NGS) has profoundly changed the approach to genetic/genomic research. Particularly, the clinical utility of NGS in detecting mutations associated with disease risk has contributed to the development of effective therapeutic strategies. Recently, comprehensive analysis of somatic genetic mutations by NGS has also been used as a new approach for controlling the quality of cell substrates for manufacturing biopharmaceuticals. However, the quality evaluation of cell substrates by NGS largely depends on the limit of detection (LOD) for rare somatic mutations. The purpose of this study was to develop a simple method for evaluating the ability of whole-exome sequencing (WES) by NGS to detect mutations with low allele frequency. To estimate the LOD of WES for low-frequency somatic mutations, we repeatedly and independently performed WES of a reference genomic DNA using the same NGS platform and assay design. LOD was defined as the allele frequency with a relative standard deviation (RSD) value of 30% and was estimated by a moving average curve of the relation between RSD and allele frequency. Results Allele frequencies of 20 mutations in the reference material that had been pre-validated by droplet digital PCR (ddPCR) were obtained from 5, 15, 30, or 40 G base pair (Gbp) sequencing data per run. There was a significant association between the allele frequencies measured by WES and those pre-validated by ddPCR, whose p-value decreased as the sequencing data size increased. By this method, the LOD of allele frequency in WES with the sequencing data of 15 Gbp or more was estimated to be between 5 and 10%. Conclusions For properly interpreting the WES data of somatic genetic mutations, it is necessary to have a cutoff threshold of low allele frequencies. The in-house LOD estimated by the simple method shown in this study provides a rationale for setting the cutoff.

scholarly journals Development of Quantitative Real-Time Polymerase Chain Reaction for Detection of and Discrimination between Erysipelothrix Rhusiopathiae and Other Erysipelothrix Species

2009 ◽  
Vol 21 (5) ◽  
pp. 701-706 ◽  
Author(s):  
Ho To ◽  
Tomohiro Koyama ◽  
Shinya Nagai ◽  
Kotaro Tuchiya ◽  
Tetsuo Nunoya
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Limit Of Detection ◽  
Enrichment Culture ◽  
Bacterial Species ◽  
Erysipelothrix Rhusiopathiae ◽  
Chain Reaction ◽  
Tissue Samples ◽  
Practical Applications ◽  
Polymerase Chain

Quantitative real-time polymerase chain reaction (qPCR) assays were developed and validated in combination with enrichment culture for the detection and discrimination of Erysipelothrix rhusiopathiae and other Erysipelothrix species from tissue samples. The targets for SYBR green qPCR assays were the 16S ribosomal RNA gene for Erysipelothrix species and a gene involved in capsular formation for E. rhusiopathiae. The specificity of the assays was assessed with Erysipelothrix species and other related bacterial species. The limit of detection was found to be 5 colony-forming units per reaction. Amplification of DNA extracted from spleen and joint samples spiked with increasing quantities of Erysipelothrix cells was shown to be equally sensitive to DNA extracted from a pure bacterial culture. The assays were evaluated with 88 tissue samples from 3 experimentally infected pigs and 50 mice and with 36 tissue samples from 3 naturally infected pigs and 11 noninfected pigs. Results were compared with those of direct qPCR and conventional culture. The qPCR after enrichment increased the diagnostic sensitivity over that of culture and qPCR, thereby significantly reducing the total time taken for the detection of E. rhusiopathiae and other Erysipelothrix species. Therefore, this technique could be used for practical applications.

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.

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