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

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%.

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A Comparative Oncology Analysis of Canine T-Cell Lymphoma (TCL): Immunohistochemical Study and Next Generation Sequencing for Protein Analysis and Genomic Target Discovery

Blood ◽

10.1182/blood.v126.23.3883.3883 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3883-3883

Author(s):

Athena Kritharis ◽

J. Tyson McDonald ◽

Afshin Beheshti ◽

Monika Pilichowska ◽

Kristine Burgess ◽

...

Keyword(s):

Dna Sequencing ◽

Sequencing Analysis ◽

Type B ◽

Next Generation ◽

Comparative Oncology ◽

Tissue Samples ◽

Next Generation Dna Sequencing ◽

Human Antibodies ◽

Network Analyses ◽

Variant Type

Abstract Background: TCLs are an uncommon, heterogeneous group of neoplasms with no consensus on optimal treatment and human 5-year survival rates <20-30%. The canine provides a potentially attractive model to study TCL in part given their spontaneously occurring cancers, intact immune system, and phylogenetic resemblance to humans. Furthermore, approximately 1/3 of all lymphomas in canine are TCL (cTCL). Previous research from our group (ASH 2014, #74755) identified 118 differentially expressed genes by RNA seq analyses comparing canine PTCL with normal canine lymph node, and PI3K, GATA3, GRB2 and PPARG as candidate biomarker genes by canonical pathway and network analyses. We aimed to further interrogate the canine as a model by histologic review and detailed genomic examination of cTCL. Methods: We evaluated de novo cTCL with immunohistochemistry (IHC) and next generation DNA sequencing for a priori genes. IHC on canine TCL was evaluated with human antibodies against CD5, CD79a, Ki67, CD3, CD4, CD8 and CD30. Canine DNA was extracted from 14 fresh frozen tissue samples and 2 paraffin embedded blocks using the QIAamp DNA Mini Kit. Utilizing the human Cancer Hotspot Panel v2 (hCHPv2), a custom expanded panel of 68 genes actively expressed in lymphoma tumor cells was created to screen cTCL for mutations. COSMIC database and PubMed was used to identify common variants expected to be present. Targets from the hCHPv2 were converted from the human genome (hg19) to positions in the canine genome (canFam3) using liftOver (UCSC Genome Browser). Following targeted amplification using the custom canine library, DNA sequencing was performed with the Ion Personal Genome Machine resulting in 4,527,638 total reads with an average length of 229 bases and 708x coverage per sample. Results: For IHC, we examined 10 primary cTCL cases utilizing human antibodies. The cTCL cases staining patterns included: 100% were CD79a negative; 80% were CD5+; Ki67 was variable; while the remaining multiple markers did not react to human antibodies. We subsequently evaluated 16 primary cTCL tumor tissue samples using DNA sequencing. There were 331 unique variants and 1474 total variants; each sample had an average of 92 variants. The most prevalent coding consequences mutations were intron variations (68%), followed by synonymous (14%) and missense variations (9%) (Fig. 1A). The most prevalent mutations were found in ATM, KIT, ERBB4, TNFAIP3, and TET2 (Fig. 1B). ATM has been implicated as a tumor suppressor and mutations have been described on a case basis in human thymic and mantle cell lymphomas, however, not in TCL. Furthermore, the majority of ATM variants identified in our analysis were non-coding or synonymous. In ATM, there were 22 mutations with 17 found as introns. The 5 coding mutations had only one of which was missense (M1758T), the others were silent (Fig. 1C). Conversely, the most prevalent variants found in the coding region were found in SMO, TP53, TNFAIP3, and TET2. Of all coding variants, 15 missense variants in TNFAIP3, JAK2, MYC, MET, SMO, DNMT3A, RB1, PIK3CA, TP53, and ERBB2 appeared to be deleterious through bioinformatics analysis. Additionally, a frameshift variant in CDH1 resulted in a 3 bp deletion that has not been described in dbSNP. Finally, mutations in KIT, TNFAIP3 and TET2 have been described in canine and human TCL with varying frequency. Conclusion: To the best of our knowledge, this represents one of the first genomic comparative oncology analyses conducted in TCL. Collectively, the DNA sequencing analysis in cTCL identified genomic similarities and novel mutations that may help unearth new oncogenic pathways in human TCL (e.g., ATM). Furthermore, the deleterious frameshift and missense mutations identified in this study as from TNFAIP3 and CDH1 are novel, of which has not been described in prior research. Continued investigation is needed towards the enhanced delineation of protein expression of cTCL and examination of the functional impact of genomic perturbations identified in cTCL in comparison to human TCL. Figure 1. Next generation DNA sequencing of canine TCL . A. Coding consequences by variant type. B. All gene variations by frequency C. Mutation map of ATM. Figure 1. Next generation DNA sequencing of canine TCL . A. Coding consequences by variant type. B. All gene variations by frequency C. Mutation map of ATM. Disclosures No relevant conflicts of interest to declare.

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The Dawn Of Next Generation DNA Sequencing In Myelodysplastic Syndromes- Experience From Pakistan.

10.21203/rs.3.rs-571430/v1 ◽

2021 ◽

Author(s):

Nida Anwar ◽

Faheem Ahmed Memon ◽

Saba Shahid ◽

Muhammad Shakeel ◽

Muhammad Irfan ◽

...

Keyword(s):

Bone Marrow ◽

Dna Sequencing ◽

Myelodysplastic Syndromes ◽

Somatic Mutations ◽

Molecular Characteristics ◽

Large Sample Size ◽

Next Generation ◽

Hematopoietic Stem ◽

Next Generation Dna Sequencing ◽

Molecular Abnormalities

Abstract Background: Myelodysplastic syndromes (MDS) are clonal disorders of hematopoietic stem cells exhibiting ineffective hematopoiesis and tendency for transformation into acute myeloid leukemia (AML). The available karyotyping and fluorescent in situ hybridization provide limited information on molecular abnormalities for diagnosis/prognosis of MDS. Next generation DNA sequencing (NGS), providing deep insights into molecular mechanisms being involved in pathophysiology, was employed to study MDS in Pakistani cohort.Patients and Methods: It was a descriptive cross-sectional study carried out at National institute of blood diseases and bone marrow transplant from 2016 to 2019. Total of 22 cases of MDS were included. Complete blood counts, bone marrow assessment and cytogenetic analysis was done. Patients were classified according to WHO classification 2016 and IPSS score was also calculated. Baseline blood samples were subjected to analysis by NGS using a panel of 54 genes associated with myeloid malignancies.Results: The median age of patients was 46 ± 15.5 years. The most common presenting complaint was weakness 10(45.45%). The mean IPSS score was 1. Cytogenetics analysis revealed abnormal karyotype in 08(36.36%) patients. On NGS, 54 non-silent rare frequency somatic mutational events in 29 genes were observed (average of 3.82 (SD ± 2.08) mutations per patient), including mutations previously not observed in MDS or AML. Notably, two genes of cohesin complex, RAD21 and STAG2, and two tumor suppressor genes, CDKN2A and TP53, contained highest number of recurrent non-silent somatic mutations in the MDS. Strikingly, a missense somatic mutation p.M272Rof Rad21 was observed in 13 cases. Overall, non-silent somatic mutations in these four genes were observed in 21 of the 22 cases. The filtration with PharmGKB database highlighted a non-synonymous genetic variant rs1042522[G > C] located in the TP53. Genotype GG and GC of this variant are associated with decreased response to cisplatin and paclitaxel chemotherapy. These two genotypes were found in 13 cases.Conclusion: Sequencing studies suggest that numerous genetic variants are involved in the initiation of MDS and in the development of AML. In countries like Pakistan where financial reservation of patients makes the use of such analysis even more difficult when the availability of advanced techniques is already a prevailing issue, our study could be an initiating effort in adding important information to the local data. Further studies and large sample size are needed in future to enlighten molecular profiling and ultimately would be helpful to compare and contrast the molecular characteristics of Asian versus global population.

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Analysis ofKRASMutations of Exon 2 Codons 12 and 13 by SNaPshot Analysis in Comparison to Common DNA Sequencing

Gastroenterology Research and Practice ◽

10.1155/2010/789363 ◽

2010 ◽

Vol 2010 ◽

pp. 1-5 ◽

Cited By ~ 16

Author(s):

Rica Zinsky ◽

Servet Bölükbas ◽

Holger Bartsch ◽

Joachim Schirren ◽

Annette Fisseler-Eckhoff

Keyword(s):

Dna Sequencing ◽

Mutation Detection ◽

Nonsmall Cell Lung Cancer ◽

Tissue Samples ◽

Exon 2 ◽

Mutation Status ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Status Analysis ◽

Formalin Fixed

Due to the call for fastKRASmutation status analysis for treatment of patients with monoclonal antibodies for metastatic colorectal cancer, sensitive, economic, and easily feasible methods are required. Under this aspect, the sensitivity and specificity of the SNaPshot analysis in comparison to the commonly used DNA sequencing was checked. We examinedKRASmutations in exon 2 codons 12 and 13 with DNA sequencing and SNaPshot analysis in 100 formalin-fixed paraffin-embedded tumor tissue samples of pancreatic carcinoma, colorectal carcinoma, and nonsmall cell lung cancer specimens of the primary tumor or metastases. 40% of these samples demonstrated mutatedKRASgenes using sequencing and SNaPshot-analysis; additional five samples (45/100) were identified only with the SNaPshot.KRASmutation detection is feasible with the reliable SNaPshot analysis method. The more frequent mutation detection by the SNaPshot analysis shows that this method has a high probability of accuracy in the detection ofKRASmutations compared to sequencing.

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Mutation detection in formalin-fixed prostate cancer biopsies taken at the time of diagnosis using next-generation DNA sequencing

Journal of Clinical Pathology ◽

10.1136/jclinpath-2014-202754 ◽

2015 ◽

Vol 68 (3) ◽

pp. 212-217 ◽

Cited By ~ 16

Author(s):

David Manson-Bahr ◽

Richard Ball ◽

Gunes Gundem ◽

Krishna Sethia ◽

Robert Mills ◽

...

Keyword(s):

Prostate Cancer ◽

Dna Sequencing ◽

Mutation Detection ◽

Next Generation ◽

Next Generation Dna Sequencing ◽

Formalin Fixed

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Faculty Opinions recommendation of Mutation detection in formalin-fixed prostate cancer biopsies taken at the time of diagnosis using next-generation DNA sequencing.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.725313523.793507587 ◽

2015 ◽

Author(s):

Hendrik van Poppel ◽

Yuri Tolkach

Keyword(s):

Prostate Cancer ◽

Dna Sequencing ◽

Mutation Detection ◽

Next Generation ◽

Next Generation Dna Sequencing ◽

Formalin Fixed

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Needlestack: an ultra-sensitive variant caller for multi-sample next generation sequencing data

10.1101/639377 ◽

2019 ◽

Cited By ~ 2

Author(s):

Tiffany M. Delhomme ◽

Patrice H. Avogbe ◽

Aurélie Gabriel ◽

Nicolas Alcala ◽

Noemie Leblay ◽

...

Keyword(s):

Next Generation Sequencing ◽

Error Rate ◽

Somatic Mutations ◽

Next Generation Sequencing Data ◽

Sequencing Error ◽

Next Generation ◽

Sequencing Error Rate ◽

Main Challenge ◽

A Genome ◽

Generation Sequencing

ABSTRACTThe emergence of Next-Generation Sequencing (NGS) has revolutionized the way of reaching a genome sequence, with the promise of potentially providing a comprehensive characterization of DNA variations. Nevertheless, detecting somatic mutations is still a difficult problem, in particular when trying to identify low abundance mutations such as subclonal mutations, tumour-derived alterations in body fluids or somatic mutations from histological normal tissue. The main challenge is to precisely distinguish between sequencing artefacts and true mutations, particularly when the latter are so rare they reach similar abundance levels as artefacts. Here, we present needlestack, a highly sensitive variant caller, which directly learns from the data the level of systematic sequencing errors to accurately call mutations. Needlestack is based on the idea that the sequencing error rate can be dynamically estimated from analyzing multiple samples together. We show that the sequencing error rate varies across alterations, illustrating the need to precisely estimate it. We evaluate the performance of needlestack for various types of variations, and we show that needlestack is robust among positions and outperforms existing state-of-the-art method for low abundance mutations. Needlestack, along with its source code is freely available on the GitHub plateform: https://github.com/IARCbioinfo/needlestack.

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

10.1101/2020.11.12.20230169 ◽

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.

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Automated high throughput nucleic acid purification from formalin-fixed paraffin-embedded tissue samples for next generation sequence analysis

PLoS ONE ◽

10.1371/journal.pone.0178706 ◽

2017 ◽

Vol 12 (6) ◽

pp. e0178706 ◽

Cited By ~ 6

Author(s):

Simon Haile ◽

Pawan Pandoh ◽

Helen McDonald ◽

Richard D. Corbett ◽

Philip Tsao ◽

...

Keyword(s):

Nucleic Acid ◽

Sequence Analysis ◽

High Throughput ◽

Next Generation ◽

Tissue Samples ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Nucleic Acid Purification ◽

Formalin Fixed

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The Dawn of next generation DNA sequencing in myelodysplastic syndromes- experience from Pakistan

BMC Genomics ◽

10.1186/s12864-021-08221-w ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Nida Anwar ◽

Faheem Ahmed Memon ◽

Saba Shahid ◽

Muhammad Shakeel ◽

Muhammad Irfan ◽

...

Keyword(s):

Bone Marrow ◽

Dna Sequencing ◽

Myelodysplastic Syndromes ◽

Somatic Mutations ◽

Molecular Characteristics ◽

Large Sample Size ◽

Next Generation ◽

Hematopoietic Stem ◽

Next Generation Dna Sequencing ◽

Molecular Abnormalities

Abstract Background Myelodysplastic syndromes (MDS) are clonal disorders of hematopoietic stem cells exhibiting ineffective hematopoiesis and tendency for transformation into acute myeloid leukemia (AML). The available karyotyping and fluorescent in situ hybridization provide limited information on molecular abnormalities for diagnosis/prognosis of MDS. Next generation DNA sequencing (NGS), providing deep insights into molecular mechanisms being involved in pathophysiology, was employed to study MDS in Pakistani cohort. Patients and methods It was a descriptive cross-sectional study carried out at National institute of blood diseases and bone marrow transplant from 2016 to 2019. Total of 22 cases of MDS were included. Complete blood counts, bone marrow assessment and cytogenetic analysis was done. Patients were classified according to revised WHO classification 2016 and IPSS score was applied for risk stratification. Baseline blood samples were subjected to analysis by NGS using a panel of 54 genes associated with myeloid malignancies. Results The median age of patients was 48.5 ± 9.19 years. The most common presenting complaint was weakness 10(45.45%). Cytogenetics analysis revealed abnormal karyotype in 10 (45.45%) patients. On NGS, 54 non-silent rare frequency somatic mutational events in 29 genes were observed (average of 3.82 (SD ± 2.08) mutations per patient), including mutations previously not observed in MDS or AML. Notably, two genes of cohesin complex, RAD21 and STAG2, and two tumor suppressor genes, CDKN2A and TP53, contained highest number of recurrent non-silent somatic mutations in the MDS. Strikingly, a missense somatic mutation p.M272Rof Rad21 was observed in 13 cases. Overall, non-silent somatic mutations in these four genes were observed in 21 of the 22 cases. The filtration with PharmGKB database highlighted a non-synonymous genetic variant rs1042522 [G > C] located in the TP53. Genotype GG and GC of this variant are associated with decreased response to cisplatin and paclitaxel chemotherapy. These two genotypes were found in 13 cases. Conclusion Sequencing studies suggest that numerous genetic variants are involved in the initiation of MDS and in the development of AML. In countries like Pakistan where financial reservation of patients makes the use of such analysis even more difficult when the availability of advanced techniques is already a prevailing issue, our study could be an initiating effort in adding important information to the local data. Further studies and large sample size are needed in future to enlighten molecular profiling and ultimately would be helpful to compare and contrast the molecular characteristics of Asian versus global population.

Download Full-text