P07.04 Rapid-CNS2: Rapid comprehensive adaptive nanopore-sequencing of CNS tumors, a proof of concept study

2021 ◽  
Vol 23 (Supplement_2) ◽  
pp. ii25-ii26
Author(s):  
H Dogan ◽  
A Patel ◽  
C Herold-Mende ◽  
S Pfister ◽  
W Wick ◽  
...  
Keyword(s):  
Copy Number ◽  
Target Selection ◽  
Turnaround Time ◽  
Diffuse Glioma ◽  
Nanopore Sequencing ◽  
Bioinformatics Pipeline ◽  
Entire Cohort ◽  
Integrated Diagnosis ◽  
Cost Efficient ◽  
Epic Array

Abstract BACKGROUND The WHO classification 2021 includes multiple molecular markers for routine diagnostics in addition to histology. Sequencing setup for complete molecular profiling requires considerable investment, while batching samples for sequencing and methylation profiling can delay turnaround time. We introduce RAPID-CNS2, a nanopore adaptive sequencing pipeline that enables comprehensive mutational, methylation and copy number profiling of CNS tumours with a single third generation sequencing assay. It can be run for single samples and offers highly flexible target selection requiring no additional library preparation. MATERIAL AND METHODS Utilising ReadFish, a toolkit enabling targeted nanopore sequencing, we sequenced DNA from 22 diffuse glioma patient samples on a MinION device. Target regions comprised our Heidelberg brain tumour NGS panel and pre-selected CpG sites for methylation classification by an adapted random forest classifier. Pathognomonic alterations, copy number profiles, and methylation classes were called using a custom bioinformatics pipeline. Results were compared to their corresponding NGS panel-seq and EPIC array outputs. RESULTS Complete concordance with the EPIC array was found for copy number profiles from RAPID-CNS2. 94% pathognomonic mutations were congruent with NGS panel-seq. MGMT promoter status was correctly identified in all samples. Methylation families were detected with 96% congruence. Among the alterations decisive for rendering a classification-compatible integrated diagnosis, 97% of the alterations were consistent over the entire cohort (completely congruent in 19/22 cases and sufficient for unequivocal diagnosis in all). CONCLUSION RAPID-CNS2 provides a swift and highly flexible alternative to conventional NGS and array-based methods for SNV/Indel analysis, detection of copy number alterations and methylation classification. The turnaround time of ~4 days can be further shortened to <12h by altering target sizes. It offers a low-capital approach that would be cost-efficient for low throughput settings and invaluable in cases requiring immediate diagnoses.

PATH-48. RAPID-CNS2: RAPID COMPREHENSIVE ADAPTIVE NANOPORE-SEQUENCING OF CNS TUMORS, A PROOF OF CONCEPT STUDY

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi126-vi126
Author(s):  
Areeba Patel ◽  
Helin Dogan ◽  
Alexander Payne ◽  
Philipp Sievers ◽  
Natalie Schoebe ◽  
...  
Keyword(s):  
Copy Number ◽  
Target Selection ◽  
Turnaround Time ◽  
Diffuse Glioma ◽  
Nanopore Sequencing ◽  
Bioinformatics Pipeline ◽  
Entire Cohort ◽  
Integrated Diagnosis ◽  
Cost Efficient ◽  
Epic Array

Abstract BACKGROUND The WHO classification 2021 includes multiple molecular markers for routine diagnostics in addition to histology. Sequencing setup for complete molecular profiling requires considerable investment, while batching samples for sequencing and methylation profiling can delay turnaround time. We introduce RAPID-CNS2, a nanopore adaptive sequencing pipeline that enables comprehensive mutational, methylation and copy number profiling of CNS tumours with a single third generation sequencing assay. It can be run for single samples and offers highly flexible target selection requiring no additional library preparation. METHODS Utilising ReadFish, a toolkit enabling targeted nanopore sequencing, we sequenced DNA from 22 diffuse glioma patient samples on a MinION device. Target regions comprised our Heidelberg brain tumour NGS panel and pre-selected CpG sites for methylation classification by an adapted random forest classifier. Pathognomonic alterations, copy number profiles, and methylation classes were called using a custom bioinformatics pipeline. Results were compared to their corresponding NGS panel-seq and EPIC array outputs. RESULTS Complete concordance with the EPIC array was found for copy number profiles from RAPID-CNS2. 94% pathognomonic mutations were congruent with NGS panel-seq. MGMT promoter status was correctly identified in all samples. Methylation families were detected with 96% congruence. Among the alterations decisive for rendering a classification-compatible integrated diagnosis, 97% of the alterations were consistent over the entire cohort (completely congruent in 19/22 cases and sufficient for unequivocal diagnosis in all). CONCLUSIONS RAPID-CNS2 provides a swift and highly flexible alternative to conventional NGS and array- based methods for SNV/Indel analysis, detection of copy number alterations and methylation classification. The turnaround time of ~4 days can be further shortened to < 12h by altering target sizes. It offers a low-capital approach that would be cost-efficient for low throughput settings and invaluable in cases requiring immediate diagnoses.

scholarly journals Rapid-CNS2: Rapid comprehensive adaptive nanopore-sequencing of CNS tumors, a proof of concept study

2021 ◽  
Author(s):  
Areeba J Patel ◽  
Helin Dogan ◽  
Alexander Payne ◽  
Philipp Sievers ◽  
Natalie Schoebe ◽  
...  
Keyword(s):  
Random Forest ◽  
Copy Number ◽  
Turnaround Time ◽  
Random Forest Classifier ◽  
Diffuse Glioma ◽  
Nanopore Sequencing ◽  
Proof Of Concept ◽  
Methylation Array ◽  
Concept Study ◽  
Integrated Diagnosis

Background: The 2021 WHO classification of central nervous system tumors includes multiple molecular markers and patterns that are recommended for routine diagnostic use in addition to histology. Sequencing infrastructures for complete molecular profiling require considerable investment, while batching samples for sequencing and methylation profiling can delay turnaround time. We introduce RAPID-CNS2, a nanopore adaptive sequencing pipeline that enables comprehensive mutational, methylation and copy number profiling of CNS tumours with a single, cost-effective sequencing assay. It can be run for single samples and offers highly flexible target selection that can be personalized per case with no additional library preparation. Methods: Utilizing ReadFish, a toolkit enabling targeted nanopore sequencing without the need for library enrichment, we sequenced DNA from 22 diffuse glioma samples on a MinION device. Target regions comprised our Heidelberg brain tumor NGS panel and pre-selected CpG sites for methylation classification using an adapted random forest classifier. Pathognomonic alterations, copy number profiles, and methylation classes were called using a custom bioinformatics pipeline. The resulting data were compared to their corresponding standard NGS panel sequencing and EPIC methylation array results. Results: Complete concordance with the EPIC array was found for copy number profiles. The vast majority (94%) of pathognomonic mutations were congruent with standard NGS panel-seq data. MGMT promoter status was correctly identified in all samples. Methylation families from the random forest classifier were detected with 96% congruence. Among the alterations decisive for rendering a WHO 2021 classification-compatible integrated diagnosis, 97% of the alterations were consistent over the entire cohort (completely congruent in 19/22 cases and sufficient for unequivocal diagnosis in all 22 samples). Conclusions: RAPID-CNS2 provides a swift and highly flexible alternative to conventional NGS and array-based methods for SNV/InDel analysis, detection of copy number alterations, target gene methylation analysis (e.g. MGMT) and methylation-based classification. The turnaround time of ~5 days for this proof-of-concept study can be further shortened to < 24h by optimizing target sizes and enabling real-time computational analysis. Expected advances in nanopore sequencing and analysis hardware make the prospect of integrative molecular diagnosis in an intra-operative setting a feasible prospect in future. This low-capital approach would be cost-efficient for low throughput settings or in locations with less sophisticated laboratory infrastructure, and invaluable in cases requiring immediate diagnoses.

scholarly journals PATH-17. INTRAGENIC COPY NUMBER BREAKPOINT ANALYSIS OF METHYLATION DATA FROM CNS TUMOURS IDENTIFIES NOVEL SUBGROUP-SPECIFIC CANDIDATE FUSION GENE ENRICHMENTS

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii427-iii428
Author(s):  
Alan Mackay ◽  
Yura Grabovska ◽  
Matthew Clarke ◽  
Diana Carvalho ◽  
Sara Temelso ◽  
...  
Keyword(s):  
Copy Number ◽  
Fusion Gene ◽  
High Grade Glioma ◽  
Structural Variations ◽  
Methylation Array ◽  
Kinase Gene ◽  
Integrated Diagnosis ◽  
Cns Tumours ◽  
Breakpoint Analysis ◽  
Balanced Translocations

Abstract Methylation array-based molecular profiling has redefined the classification of brain tumours and now forms an important part of their integrated diagnosis, providing both subgroup assignment and genome wide DNA copy number profiles. These latter data can be used to identify intragenic breakpoints which are frequently associated with structural variations resulting in therapeutically targetable oncogenic fusion genes. To systematically assess the landscape of these alterations, we combined publicly available methylation datasets resulting in a total of 5660 CNS tumours, around half paediatric, and including &gt;1000 high grade glioma and DIPG. These were analysed by standard methodology (MNP, conumee), and intragenic breakpoint enrichment was compared within methylation subgroups, superfamilies, and tumours with no high-scoring classification. Benchmarking included sequence-verified cases such as infant hemispheric gliomas (IHG) with ALK(15%) and ROS1(7%) fusions, and pathognomic alterations associated with specific entities such as RELA-EPN, MYB-LGG and HGNET-MN1. We identified previously unreported enrichments of well-recognised fusion targets such as NTRK2in GBM_MID and NTRK3in DMG_K27 (both 5%), METin A_IDH / A_IDH_HG (3–5%), and FGFR1/3in GBM_G34 (8–9%). Novel recurrent kinase gene candidates to be verified and explored further include IGF1Rin 2–12% cases spanning glioma subgroups, and TIE1in poorly classified tumours. This latter ‘NOS’ group were also enriched in various transcription factor targets of breakpoints, including TCF4and PLAGL2. Despite limitations due to sample quality, resolution or balanced translocations, breakpoint analysis of methylation copy number profiles provides simple screening for structural rearrangements which may directly influence targeted therapy in paediatric CNS tumours.

Novel Recurrent Altered Genes in Chinese Patients With Anaplastic Thyroid Cancer

2021 ◽  
Author(s):  
Lingyun Zhang ◽  
Zhixiang Ren ◽  
Zhengzheng Su ◽  
Yang Liu ◽  
Tian Yang ◽  
...  
Keyword(s):  
Thyroid Cancer ◽  
Copy Number ◽  
Treatment Strategies ◽  
Gene Mutations ◽  
Anaplastic Thyroid Cancer ◽  
Chinese Patients ◽  
Driver Gene ◽  
Phosphatidylinositol 3 Kinase ◽  
Entire Cohort ◽  
Whole Exome

Abstract Background Anaplastic thyroid cancer (ATC) is a rare but lethal malignancy, and few systematic investigations on genomic profiles of ATC have been performed in Chinese patients. Methods Fifty-four ATC patients in West China Hospital between 2010 to 2020 were retrospectively analyzed, while 29 patients with available samples were sequenced by whole-exome sequencing (WES). The associations between genomic alterations and clinical characteristics were statistically evaluated. Results The median overall survival was 3.0 months in the entire cohort, which was impacted by multiple clinical features, including age, tumor size, and different treatment strategies. In the WES cohort, totally 797 nonsilent mutations were detected; the most frequently altered genes were TP53 (48%), BRAF (24%), PIK3CA (24%), and TERT promoter (21%). Although these mutations have been well-reported in previous studies, ethnic specificity was exhibited in terms of mutation frequency. Moreover, several novel significantly mutated genes were identified including RBM15 (17%), NOTCH2NL (14%), CTNNA3 (10%), and KATNAL2 (10%). WES-based copy number alteration analysis also revealed a high frequent gain of NOTCH2NL (41%), which induced its increased expression. Gene mutations and copy number alterations were enriched in phosphatidylinositol 3-kinase/AKT/mechanistic target of rapamycin (mTOR), NOTCH, and WNT pathways. Conclusions This study reveals shared and ethnicity-specific genomic profiles of ATC in Chinese patients and suggests NOTCH2NL may act as a novel candidate driver gene for ATC tumorigenesis.

scholarly journals High resolution copy number inference in cancer using short-molecule nanopore sequencing

2020 ◽  
Author(s):  
Timour Baslan ◽  
Sam Kovaka ◽  
Fritz J. Sedlazeck ◽  
Yanming Zhang ◽  
Robert Wappel ◽  
...  
Keyword(s):  
Copy Number ◽  
Cost Effective ◽  
Chromosome Analysis ◽  
Ease Of Use ◽  
Precision Oncology ◽  
Nanopore Sequencing ◽  
Dna Molecules ◽  
Sequencing Data ◽  
Short Read ◽  
Short Read Sequencing

ABSTRACTGenome copy number is an important source of genetic variation in health and disease. In cancer, clinically actionable Copy Number Alterations (CNAs) can be inferred from short-read sequencing data, enabling genomics-based precision oncology. Emerging Nanopore sequencing technologies offer the potential for broader clinical utility, for example in smaller hospitals, due to lower instrument cost, higher portability, and ease of use. Nonetheless, Nanopore sequencing devices are limited in terms of the number of retrievable sequencing reads/molecules compared to short-read sequencing platforms. This represents a challenge for applications that require high read counts such as CNA inference. To address this limitation, we targeted the sequencing of short-length DNA molecules loaded at optimized concentration in an effort to increase sequence read/molecule yield from a single nanopore run. We show that sequencing short DNA molecules reproducibly returns high read counts and allows high quality CNA inference. We demonstrate the clinical relevance of this approach by accurately inferring CNAs in acute myeloid leukemia samples. The data shows that, compared to traditional approaches such as chromosome analysis/cytogenetics, short molecule nanopore sequencing returns more sensitive, accurate copy number information in a cost effective and expeditious manner, including for multiplex samples. Our results provide a framework for the sequencing of relatively short DNA molecules on nanopore devices with applications in research and medicine, that include but are not limited to, CNAs.

scholarly journals Third-Generation Sequencing in the Clinical Laboratory: Exploring the Advantages and Challenges of Nanopore Sequencing

2019 ◽  
Vol 58 (1) ◽  
Author(s):  
Lauren M. Petersen ◽  
Isabella W. Martin ◽  
Wayne E. Moschetti ◽  
Colleen M. Kershaw ◽  
Gregory J. Tsongalis
Keyword(s):  
Infectious Disease ◽  
Pathogen Detection ◽  
Clinical Laboratory ◽  
Low Cost ◽  
Turnaround Time ◽  
Metagenomic Sequencing ◽  
Nanopore Sequencing ◽  
Advantages And Disadvantages ◽  
Disease Diagnostics ◽  
Infectious Disease Diagnostics

ABSTRACT Metagenomic sequencing for infectious disease diagnostics is an important tool that holds promise for use in the clinical laboratory. Challenges for implementation so far include high cost, the length of time to results, and the need for technical and bioinformatics expertise. However, the recent technological innovation of nanopore sequencing from Oxford Nanopore Technologies (ONT) has the potential to address these challenges. ONT sequencing is an attractive platform for clinical laboratories to adopt due to its low cost, rapid turnaround time, and user-friendly bioinformatics pipelines. However, this method still faces the problem of base-calling accuracy compared to other platforms. This review highlights the general challenges of pathogen detection in clinical specimens by metagenomic sequencing, the advantages and disadvantages of the ONT platform, and how research to date supports the potential future use of nanopore sequencing in infectious disease diagnostics.

scholarly journals A Sample-to-Report Solution for Taxonomic Identification of Cultured Bacteria in the Clinical Setting Based on Nanopore Sequencing

2020 ◽  
Vol 58 (6) ◽  
Author(s):  
Stefan Moritz Neuenschwander ◽  
Miguel Angel Terrazos Miani ◽  
Heiko Amlang ◽  
Carmen Perroulaz ◽  
Pascal Bittel ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Sequence Data ◽  
Automated Analysis ◽  
Amplicon Sequencing ◽  
Turnaround Time ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Nanopore Sequencing ◽  
Taxonomic Identification ◽  
Consensus Analysis

ABSTRACT Amplicon sequencing of the 16S rRNA gene is commonly used for the identification of bacterial isolates in diagnostic laboratories and mostly relies on the Sanger sequencing method. The latter, however, suffers from a number of limitations, with the most significant being the inability to resolve mixed amplicons when closely related species are coamplified from a mixed culture. This often leads to either increased turnaround time or absence of usable sequence data. Short-read next-generation sequencing (NGS) technologies could solve the mixed amplicon issue but would lack both cost efficiency at low throughput and fast turnaround times. Nanopore sequencing developed by Oxford Nanopore Technologies (ONT) could solve those issues by enabling a flexible number of samples per run and an adjustable sequencing time. Here, we report on the development of a standardized laboratory workflow combined with a fully automated analysis pipeline LORCAN (long read consensus analysis), which together provide a sample-to-report solution for amplicon sequencing and taxonomic identification of the resulting consensus sequences. Validation of the approach was conducted on a panel of reference strains and on clinical samples consisting of single or mixed rRNA amplicons associated with various bacterial genera by direct comparison to the corresponding Sanger sequences. Additionally, simulated read and amplicon mixtures were used to assess LORCAN’s behavior when dealing with samples with known cross-contamination levels. We demonstrate that by combining ONT amplicon sequencing results with LORCAN, the accuracy of Sanger sequencing can be closely matched (>99.6% sequence identity) and that mixed samples can be resolved at the single-base resolution level. The presented approach has the potential to significantly improve the flexibility, reliability, and availability of amplicon sequencing in diagnostic settings.

scholarly journals An Intelligent and Cost-Efficient Resource Consolidation Algorithm in Nanoscale Computing Environments

2020 ◽  
Vol 10 (18) ◽  
pp. 6494
Author(s):  
MeSuk Kim ◽  
ALam Han ◽  
TaeYoung Kim ◽  
JongBeom Lim
Keyword(s):  
Fog Computing ◽  
Turnaround Time ◽  
Nanoscale Devices ◽  
Efficient Resource ◽  
Computing Environments ◽  
Cost Efficient ◽  
Cloud Users ◽  
Resource Consolidation ◽  
The Internet Of Things ◽  
Cloud User

Because the Internet of things (IoT) and fog computing are prevalent, an efficient resource consolidation scheme in nanoscale computing environments is urgently needed. In nanoscale environments, a great many small devices collaborate to achieve a predefined goal. The representative case would be the edge cloud, where small computing servers are deployed close to the cloud users to enhance the responsiveness and reduce turnaround time. In this paper, we propose an intelligent and cost-efficient resource consolidation algorithm in nanoscale computing environments. The proposed algorithm is designed to predict nanoscale devices’ scheduling decisions and perform the resource consolidation that reconfigures cloud resources dynamically when needed without interrupting and disconnecting the cloud user. Because of the large number of nanoscale devices in the system, we developed an efficient resource consolidation algorithm in terms of complexity and employed the hidden Markov model to predict the devices’ scheduling decision. The performance evaluation shows that our resource consolidation algorithm is effective for predicting the devices’ scheduling decisions and efficiency in terms of overhead cost and complexity.

scholarly journals A Simple, Universal, and Cost-Efficient Digital PCR Method for the Targeted Analysis of Copy Number Variations

2019 ◽  
Vol 65 (9) ◽  
pp. 1153-1160 ◽  
Author(s):  
Kévin Cassinari ◽  
Olivier Quenez ◽  
Géraldine Joly-Hélas ◽  
Ludivine Beaussire ◽  
Nathalie Le Meur ◽  
...  
Keyword(s):  
Copy Number ◽  
Segregation Analysis ◽  
Genetic Diseases ◽  
Genomic Medicine ◽  
Digital Pcr ◽  
Copy Number Variations ◽  
Locked Nucleic Acid ◽  
Targeted Analysis ◽  
Cost Efficient ◽  
High Flexibility

Abstract BACKGROUND Rare copy number variations (CNVs) are a major cause of genetic diseases. Simple targeted methods are required for their confirmation and segregation analysis. We developed a simple and universal CNV assay based on digital PCR (dPCR) and universal locked nucleic acid (LNA) hydrolysis probes. METHODS We analyzed the mapping of the 90 LNA hydrolysis probes from the Roche Universal ProbeLibrary (UPL). For each CNV, selection of the optimal primers and LNA probe was almost automated; probes were reused across assays and each dPCR assay included the CNV amplicon and a reference amplicon. We assessed the assay performance on 93 small and large CNVs and performed a comparative cost-efficiency analysis. RESULTS UPL-LNA probes presented nearly 20000000 occurrences on the human genome and were homogeneously distributed with a mean interval of 156 bp. The assay accurately detected all the 93 CNVs, except one (&lt;200 bp), with coefficient of variation &lt;10%. The assay was more cost-efficient than all the other methods. CONCLUSIONS The universal dPCR CNV assay is simple, robust, and cost-efficient because it combines a straightforward design allowed by universal probes and end point PCR, the advantages of a relative quantification of the target to the reference within the same reaction, and the high flexibility of the LNA hydrolysis probes. This method should be a useful tool for genomic medicine, which requires simple methods for the interpretation and segregation analysis of genomic variations.

Sign in / Sign up

Export Citation Format

Share Document