The study was supported by the Italian Cancer Genome Project (Grant No. FIRB RBAP10AHJB), Associazione Italiana Ricerca Cancro (AIRC; Grant No. 12182 to AS and 18178 to VC), FP7 European Community Grant (Cam-Pac No 602783 to AS). The funding agencies had no role in the collection, analysis and interpretation of data or in the writing of the manuscript.

<ol><li>Koboldt, D. C., Steinberg, K. M., Larson, D. E., Wilson, R. K., Mardis, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+next-generation+sequencing+revolution+and+its+impact+on+genomics%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">The next-generation sequencing revolution and its impact on genomics.</a> Cell. 155 (1), 27-38 (2013).</li>
<li>McGranahan, N., Swanton, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clonal+Heterogeneity+and+Tumor+Evolution%3A+Past%2C+Present%2C+and+the+Future%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Clonal Heterogeneity and Tumor Evolution: Past, Present, and the Future.</a> Cell. 168 (4), 613-628 (2017).</li>
<li>Stratton, M. R., Campbell, P. J., Futreal, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+cancer+genome%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">The cancer genome.</a> Nature. 458 (7239), 719-724 (2009).</li>
<li>Makohon-Moore, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Limited+heterogeneity+of+known+driver+gene+mutations+among+the+metastases+of+individual+patients+with+pancreatic+cancer%5BTitle%5D)+AND+%22Nature+Genetics%22%5BJournal%5D)">Limited heterogeneity of known driver gene mutations among the metastases of individual patients with pancreatic cancer.</a> Nature Genetics. 49 (3), 358-366 (2017).</li>
<li>Seyfried, T. N., Huysentruyt, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((On+the+origin+of+cancer+metastasis%5BTitle%5D)+AND+%22Critical+reviews+in+oncogenesis%22%5BJournal%5D)">On the origin of cancer metastasis.</a> Critical reviews in oncogenesis. 18 (1-2), 43-73 (2013).</li>
<li>McLaren, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Deriving+the+consequences+of+genomic+variants+with+the+Ensembl+API+and+SNP+Effect+Predictor%5BTitle%5D)+AND+%22Bioinformatics%22%5BJournal%5D)">Deriving the consequences of genomic variants with the Ensembl API and SNP Effect Predictor.</a> Bioinformatics. 26 (16), 2069-2070 (2010).</li>
<li>Robinson, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integrative+genomics+viewer%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">Integrative genomics viewer.</a> Nature Biotechnology. 29 (1), 24-26 (2011).</li>
<li>Amato, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+alterations+associated+with+metastases+of+solid+pseudopapillary+neoplasms+of+the+pancreas%5BTitle%5D)+AND+%22The+Journal+of+Pathology%22%5BJournal%5D)">Molecular alterations associated with metastases of solid pseudopapillary neoplasms of the pancreas.</a> The Journal of Pathology. 247 (1), 123-134 (2019).</li>
<li>Mafficini, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reporting+tumor+molecular+heterogeneity+in+histopathological+diagnosis%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Reporting tumor molecular heterogeneity in histopathological diagnosis.</a> PLoS One. 9 (8), e104979(2014).</li>
<li>Shi, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reliability+of+Whole-Exome+Sequencing+for+Assessing+Intratumor+Genetic+Heterogeneity%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">Reliability of Whole-Exome Sequencing for Assessing Intratumor Genetic Heterogeneity.</a> Cell Reports. 25 (6), 1446-1457 (2018).</li>
<li>Simbolo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DNA+qualification+workflow+for+next+generation+sequencing+of+histopathological+samples%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">DNA qualification workflow for next generation sequencing of histopathological samples.</a> PLoS One. 8 (6), e62692(2013).</li>
<li>Connor, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integration+of+Genomic+and+Transcriptional+Features+in+Pancreatic+Cancer+Reveals+Increased+Cell+Cycle+Progression+in+Metastases%5BTitle%5D)+AND+%22Cancer+Cell%22%5BJournal%5D)">Integration of Genomic and Transcriptional Features in Pancreatic Cancer Reveals Increased Cell Cycle Progression in Metastases.</a> Cancer Cell. 35 (2), 267-282 (2019).</li>
<li>Priestley, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pan-cancer+whole+genome+analyses+of+metastatic+solid+tumors%5BTitle%5D)+AND+%22bioRxiv%22%5BJournal%5D)">Pan-cancer whole genome analyses of metastatic solid tumors.</a> bioRxiv. , (2018).</li>
</ol>

The authors have nothing to disclose.

Our method enables the identification of molecular alterations involved in progression of solid tumors through integration of vertical data (i.e., morphology, DNA sequencing, and immunohistochemistry) from distinct lesions of a given patient. We demonstrated the capability of our method to detect clonal and subclonal events in a mutational silent tumor type (i.e., SPN, solid-pseudopapillary neoplasm of the pancreas) by interrogating the coding sequences of 409 cancer relevant genes8. An advantage ...

The advent of next generation sequencing (NGS) has revolutionized the way cancers are diagnosed and treated1. NGS coupled to multiregional sequencing have exposed a high degree of intra-tumoral heterogeneity (ITH) in solid tumors2, which explains in part the failure of targeted therapy due to the presence of subclones with differing drug sensitivity2. An important challenge posed by genome-wide sequencing studies is the necessity to distinguish between passenger (i.e., neutral) and driver mutations in individual cancers3. Several studies have indeed shown that, in certain tumors, passenger mutations account for the majority of ITH, while driver alterations tend to be conserved among lesions of the same individual4. It is also important to note that large mutational burden (as seen in lung cancers and melanoma) does not necessarily imply a large subclonal mutational burden2. Therefore, a high degree of ITH can be found in tumors with low mutational burden.
Metastases are responsible for more than 90% of cancer-related death worldwide5; therefore, capturing the mutational heterogeneity of driver genes among primary and metastatic lesions is critical to the design of effective therapies for advanced-stage diseases. Clinical sequencing is generally performed on nucleic acids from fixed tissues, which renders genome-wide exploration difficult because of poor DNA quality. On the other hand, the intent of clinical sequencing is to identify actionable mutations and/or mutations that might predict responsiveness/unresponsiveness to a given therapeutic regimen. As it stands, sequencing can be restricted to a smaller fraction of the genome for timely extraction of clinically relevant information. The transition from low-throughput DNA profiling (e.g., Sanger Sequencing) to NGS has rendered it possible to analyze hundreds of cancer-relevant genes at a high depth of coverage, which allows for the detection of subclonal events. Here, we report a method for comparative lesions analysis that allows for the identification of clonal and subclonal populations among different specimens from the same individual. The method described here integrates three well-established approaches (histological analysis, high-coverage multi-lesion sequencing, and immunophenotypic analyses) to predict functional consequences of the variations identified. The approach is schematically described in Figure 1 and has been applied to the study of 5 metastatic cases of solid pseudopapillary neoplasms (SPNs) of the pancreas. While we describe processing and analysis of formalin-fixed paraffin-embedded (FFPE) tissue specimens, the same procedure can be applied to genetic material from fresh-frozen tissue.

<table><tbody><tr><td>2100 Bioanalyzer Instrument</td><td>Agilent Technologies</td><td>G2939BA</td><td>&amp;nbsp;Automated electrophoresis tool</td></tr><tr><td>Agencourt AMPure XP Kit</td><td>Fisher Scientific</td><td>NC9959336</td><td>Beads technology for the purification of PCR products; beads-based purification reagent</td></tr><tr><td>Agilent High Sensitivity DNA Kit</td><td>Agilent Technologies</td><td>5067-4627</td><td>Library quantification</td></tr><tr><td>Anti-BAP1</td><td>Santa Cruz Biotechnology</td><td>sc-28383</td><td>Antibody</td></tr><tr><td>Anti-GLUT1</td><td>Thermo Scientific</td><td>RB-9052</td><td>Antibody</td></tr><tr><td>Anti-KDM6A</td><td>Cell Signaling</td><td>#33510</td><td>Antibody</td></tr><tr><td>Anti-p53</td><td>Novocastra</td><td>NCL-L-p53-DO7</td><td>Antibody</td></tr><tr><td>Anti-&amp;beta;catenin</td><td>Sigma-Aldrich</td><td>C7207</td><td>Antibody</td></tr><tr><td>Blocking Solution</td><td>home made</td><td>-</td><td>5 % Bovine serum albumin (BSA) in TBST</td></tr><tr><td>Endogenous peroxidases inactivation solution</td><td>home made</td><td>-</td><td>3% H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; in Tris-buffered saline (TBS) 1x</td></tr><tr><td>Leica CV ultra</td><td>Leica</td><td>70937891</td><td>Entellan mountin media</td></tr><tr><td>Epitope Retrieval Solution 1</td><td>Leica Biosystems</td><td>AR9961</td><td>Citrate based pH 6.0 epitope retrieval solution</td></tr><tr><td>Epitope Retrieval Solution 2</td><td>Leica Biosystems</td><td>AR9640</td><td>EDTA based pH 9.0 epitope retrieval solution</td></tr><tr><td>Eppendorf 0.2 ml PCR Tubes, clear</td><td>Eppendorf</td><td>951010006</td><td>Tubes</td></tr><tr><td>Eppendorf DNA LoBind Tubes, 1.5 mL</td><td>Eppendorf</td><td>22431021</td><td>Tubes</td></tr><tr><td>Ethanol</td><td>DIAPATH</td><td>A0123</td><td>IHC deparaffinization reagent</td></tr><tr><td>ImmEdge Pen&amp;nbsp;Hydrophobic&amp;nbsp;Barrier&amp;nbsp;Pen</td><td>Vector Laboratories</td><td>H&amp;shy;4000</td><td>Hydrophobic Pen</td></tr><tr><td>ImmPACT DAB&amp;nbsp;Peroxidase</td><td>Vector Laboratories</td><td>SK&amp;shy;4105</td><td>HRP substrate</td></tr><tr><td>ImmPRESS Anti&amp;shy;Rabbit&amp;nbsp;Ig&amp;nbsp;Reagent&amp;nbsp;Peroxidase</td><td>Vector Laboratories</td><td>MP&amp;shy;7401&amp;shy;50</td><td>Secondary antibody</td></tr><tr><td>ImmPRESS Anti&amp;shy;Mouse&amp;nbsp;Ig&amp;nbsp;Reagent&amp;nbsp;Peroxidase</td><td>Vector Laboratories</td><td>MP&amp;shy;7402&amp;shy;50</td><td>Secondary antibody</td></tr><tr><td>Integrative Genomics Viewer (IGV)</td><td>Broad Institute</td><td>-</td><td>https://software.broadinstitute.org/software/igv/home</td></tr><tr><td>Ion AmpliSeq Comprehensive Cancer Panel</td><td>Thermofisher Scientific</td><td>4477685</td><td>Multiplexed target selection of 409 cancer related gene. https://assets.thermofisher.com/TFS-Assets/CSD/Reference-Materials/ion-ampliseq-cancer-panel-gene-list.pdf</td></tr><tr><td>Ion AmpliSeq Library Kit 2.0</td><td>Thermofisher Scientific</td><td>4480441</td><td>Preparation of amplicon libraries using Ion AmpliSeq panels</td></tr><tr><td>Ion Chef Instrument</td><td>Thermofisher Scientific</td><td>4484177</td><td>Automated library preparation, template preparation and chip loading</td></tr><tr><td>Ion PI Chip Kit v3 or Ion 540 Chip</td><td>Thermofisher Scientific</td><td>A26771 or A27766</td><td>Barcoded chips for sequencing</td></tr><tr><td>Ion PI Hi-Q Chef Kit or Ion 540 Kit-Chef</td><td>Thermofisher Scientific</td><td>A27198 or A30011</td><td>Template preparation</td></tr><tr><td>Ion PI Hi-Q Sequencing 200 Kit or Ion S5 Sequencing Kit</td><td>Thermofisher Scientific</td><td>A26433 or A30011</td><td>Sequencing</td></tr><tr><td>Ion Proton or Ion GeneStudio S5 System</td><td>Thermofisher Scientific</td><td>4476610 or A38196</td><td>Sequencing system</td></tr><tr><td>Ion Reporter Software - AmpliSeq Comprehensive Cancer Panel tumour-normal pair</td><td>Thermofisher Scientific</td><td>4487118</td><td>Workflow</td></tr><tr><td>Ion Reporter Software - uploader plugin</td><td>Thermofisher Scientific</td><td>4487118</td><td>Data analysis tool</td></tr><tr><td>Ion Torrent Suite Software - Coverege Analysis plugin</td><td>Thermofisher Scientific</td><td>4483643</td><td>Plugin that describe the level of sequance coverage produced</td></tr><tr><td>Ion Torrent Suite Software - Variant Caller plugin</td><td>Thermofisher Scientific</td><td>4483643</td><td>Plugin able to identify single-nucleotide polymorphisms (SNPs), insertions and deletions in a sample across a reference</td></tr><tr><td>Ion Xpress Barcode Adapters 1-96 Kit</td><td>Thermofisher Scientific</td><td>4474517</td><td>Unique barcode adapters</td></tr><tr><td>NanoDrop 2000/2000c Spectrophotometers</td><td>Thermofisher Scientific</td><td>ND-2000</td><td>DNA purity detection</td></tr><tr><td>NCBI reference sequence (RefSeq) database</td><td>NCBI</td><td>-</td><td>https://www.ncbi.nlm.nih.gov/refseq/</td></tr><tr><td>Platinum PCR SuperMix High Fidelity</td><td>Fisher Scientific</td><td>12532-016 or 12532-024</td><td>SuperMix for PCR amplification; high-fidelity PCR mix</td></tr><tr><td>QIAamp DNA Blood Mini Kit</td><td>Quiagen</td><td>51106 0r 51104</td><td>DNA blood extraction kit</td></tr><tr><td>QIAamp DNA FFPE Tissue</td><td>Quiagen</td><td>56404</td><td>DNA FFPE tissue extraction kit</td></tr><tr><td>Qubit 2.0 Fluorometer</td><td>Thermofisher Scientific</td><td>Q32866</td><td>DNA quantification</td></tr><tr><td>Qubit dsDNA BR Assay Kit</td><td>Thermofisher Scientific</td><td>Q32850</td><td>Kit for DNA quantification on Qubit 2.0 Fluorometer</td></tr><tr><td>TBST</td><td>home made</td><td>-</td><td>Tris-buffered saline (TBS) and 0.1% of Tween 20</td></tr><tr><td>Tissue-Tek Prisma Plus &amp;amp; Tissue-Tek Film</td><td>Sakura Europe</td><td>6172</td><td>Automated tissue slide stainer instrument</td></tr><tr><td>Variant Effect Predictor (VEP) software</td><td>EMBI-EBI</td><td>-</td><td>http://grch37.ensembl.org/Homo_sapiens /Tools/VEP</td></tr><tr><td>Xilene, mix of isomeres</td><td>Carlo Erba</td><td>492306</td><td>IHC deparaffinization reagent</td></tr></tbody></table>

comparative lesions analysis through a targeted sequencing approach

Assessing intra-tumoral heterogeneity (ITH) is of paramount importance to anticipate failure of targeted therapies and design accordingly effective anti-tumor strategies. Although concerns are frequently raised due to differences in sample processing and depth of coverage, next-generation sequencing of solid tumors have unraveled a highly variable degree of ITH across tumor types. Capturing the genetic relatedness between primary and metastatic lesions through the identification of clonal and subclonal populations is critical to the design of therapies for advance-stage diseases. Here, we report a method for comparative lesions analysis that allows for the identification of clonal and subclonal populations among different specimens from the same patient. The experimental approach described here integrates three well-established approaches: histological analysis, high-coverage multi-lesion sequencing, and immunophenotypic analyses. In order to minimize the effects on the detection of subclonal events by inappropriate sample processing, we subjected tissues to careful pathological examination and neoplastic cell enrichment. Quality controlled DNA from neoplastic lesions and normal tissues was then subjected to high coverage sequencing, targeting the coding regions of 409 relevant cancer genes. While only looking at a limited genomic space, our approach enables evaluating the extent of heterogeneity among somatic alterations (single-nucleotide mutations and copy-number variations) in distinct lesions from a given patient. Through comparative analysis of sequencing data, we were able to distinguish clonal vs. subclonal alterations. The majority of ITH is often ascribed to passenger mutations; therefore, we also used immunohistochemistry to predict functional consequences of mutations. While this protocol has been applied to a specific tumor type, we anticipate that the methodology described here is broadly applicable to other solid tumor types.

The study workflow is illustrated in Figure 1. Multi-lesions (n = 13) sequencing of 5 SPN cases targeting the coding sequences of 409 cancer related genes identified a total of 27 somatic mutations in 8 genes (CTNNB1, KDM6A, BAP1, TET1, SMAD4, TP53, FLT1, and FGFR3). Mutations were defined as founder/clonal when shared among all lesions of a given patient, and progressor/subclonal when detected in some but not all lesion...

Watch this Scientific Journal Video about Comparative Lesions Analysis Through a Targeted Sequencing Approach at JoVE.com

Comparative Lesions Analysis Through a Targeted Sequencing Approach

This article describes a method to identify clonal and subclonal alterations among different specimens from a given patient. Although the experiments described here focus on a specific tumor type, the approach is broadly applicable to other solid tumors.

Assessing intra-tumoral heterogeneity (ITH) is of paramount importance to anticipate failure of targeted therapies and design accordingly effective ...

comparative-lesions-analysis-through-a-targeted-sequencing-approach

Research

JoVE Journal

Behavior

6.7K Views.  University and Hospital Trust of Verona. Ons protocol maakt het vastleggen van de genetische verwantheid tussen primaire en gemetastaseerdlaesies mogelijk door de identificatie van moleculaire veranderingen die betrokken zijn bij ziekteprogressie. Onze methode biedt de mogelijkheid om genetische verwantschap in klinisch monster met een hoge gevoeligheid en specificiteit te verkennen. Dit is te wijten aan de integratie van moleculaire en histopathologische analyse.De beoordeling van intratumor heterogeniteit in het kankeronder...

Video: Comparative Lesions Analysis Through a Targeted Sequencing Approach

Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific molecular profiles of a tumor may predict responsiveness to therapeutic agents or provide knowledge about prognosis. At our institution a tumor genotyping program was established as part of routine clinical care, screening both hematologic and solid tumors for a wide spectrum of mutations using two next-generation sequencing (NGS) panels: a custom, 33 gene hematological malignancies panel for use with peripheral blood and bone marrow, and a commercially produced solid tumor panel for use with formalin-fixed paraffin-embedded tissue that targets 47 genes commonly mutated in cancer. Our workflow includes a pathologist review of the biopsy to ensure there is adequate amount of tumor for the assay followed by customized DNA extraction is performed on the specimen. Quality control of the specimen includes steps for quantity, quality and integrity and only after the extracted DNA passes these metrics an amplicon library is generated and sequenced. The resulting data is analyzed through an in-house bioinformatics pipeline and the variants are reviewed and interpreted for pathogenicity. Here we provide a snapshot of the utility of each panel using two clinical cases to provide insight into how a well-designed NGS workflow can contribute to optimizing clinical outcomes.

This manuscript describes clinical protocols for two next-generation sequencing panels. One panel interrogates hematologic malignancies while the other panel targets genes commonly mutated in solid tumors. Molecular classification of driver mutations in human malignancies offers valuable prognostic and predictive information.

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific ...

Cancer Research

Flow-sorting and Exome Sequencing of the Reed-Sternberg Cells of Classical Hodgkin Lymphoma

The Hodgkin Reed-Sternberg cells of classical Hodgkin lymphoma are sparsely distributed within a background of inflammatory lymphocytes and typically comprise less than 1% of the tumor mass. Material derived from bulk tumor contains tumor content at a concentration insufficient for characterization. Therefore, fluorescence activated cell sorting using eight antibodies, as well as side- and forward-scatter, is described here as a method of rapidly separating and concentrating with high purity thousands of HRS cells from the tumor for subsequent study. At the same time, because standard protocols for exome sequencing typically require 100-1,000 ng of input DNA, which is often too high, even with flow sorting, we also provide an optimized, low-input library construction protocol capable of producing high-quality data from as little as 10 ng of input DNA. This combination is capable of producing next-generation libraries suitable for hybridization capture of whole-exome baits or more focused targeted panels, as desired. Exome sequencing of the HRS cells, when compared against healthy intratumor T or B cells, can identify somatic alterations, including mutations, insertions and deletions, and copy number alterations. These findings elucidate the molecular biology of HRS cells and may reveal avenues for targeted drug treatments.

Here, we describe a combined flow cytometric cell sorting and low-input, next-generation library construction protocol designed to produce high-quality, whole-exome data from the Hodgkin Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (CHL).

The Hodgkin Reed-Sternberg cells of classical Hodgkin lymphoma are sparsely distributed within a background of inflammatory lymphocytes and typically ...

Genetics

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissues are widely used for genetic testing. However, FFPE DNA is generally damaged and fragmented during the fixation process with formalin. Therefore, FFPE DNA is sometimes not adequate for genetic testing because of low quality and quantity of DNA. Here we present a method of touch imprint cytology (TIC) to obtain genomic DNA from cancer cells, which can be observed under a microscope. Cell morphology and cancer cell numbers can be evaluated using TIC specimens. Furthermore, the extraction of genomic DNA from TIC samples can be completed within two days. The total amount and quality of TIC DNA obtained using this method was higher than that of FFPE DNA. This rapid and simple method allows researchers to obtain high-quality DNA for genetic testing (e.g., next generation sequencing analysis, digital PCR, and quantitative real time PCR) and to shorten the turnaround time for reporting results.

Obtaining high-quality genomic DNA from tumor tissues is an essential first step for analyzing genetic alterations using next generation sequencing. In this article, we present a simple and rapid method to enrich tumor cells and obtain intact DNA from touch imprint cytology specimens.

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The technique is highly efficient with millions of sequencing reads being produced in a short time span and at relatively low cost. Specifically, targeted NGS is able to focus investigations to genomic regions of particular interest based on the disease of study. Not only does this further reduce costs and increase the speed of the process, but it lessens the computational burden that often accompanies NGS. Although targeted NGS is restricted to certain regions of the genome, preventing identification of potential novel loci of interest, it can be an excellent technique when faced with a phenotypically and genetically heterogeneous disease, for which there are previously known genetic associations. Because of the complex nature of the sequencing technique, it is important to closely adhere to protocols and methodologies in order to achieve sequencing reads of high coverage and quality. Further, once sequencing reads are obtained, a sophisticated bioinformatics workflow is utilized to accurately map reads to a reference genome, to call variants, and to ensure the variants pass quality metrics. Variants must also be annotated and curated based on their clinical significance, which can be standardized by applying the American College of Medical Genetics and Genomics Pathogenicity Guidelines. The methods presented herein will display the steps involved in generating and analyzing NGS data from a targeted sequencing panel, using the ONDRISeq neurodegenerative disease panel as a model, to identify variants that may be of clinical significance.

Targeted next-generation sequencing is a time- and cost-efficient approach that is becoming increasingly popular in both disease research and clinical diagnostics. The protocol described here presents the complex workflow required for sequencing and the bioinformatics process used to identify genetic variants that contribute to disease.

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The ...

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic target analyses and drug response monitoring using patient derived xenografts (PDX). This strategy was validated using ovarian tumors as an example. The mate-pair next generation sequencing (MPseq) protocol was used to identify structural alterations and followed by analysis of potentially targetable alterations. Human tumors grown in immunocompromised mice were treated with drugs selected based on the genomic analyses. Results demonstrated a good correlation between the predicted and the observed responses in the PDX model. The presented approach can be used to test the efficacy of combination treatments and aid personalized treatment for patients with recurrent cancer, specifically in cases when standard therapy fails and there is a need to use drugs off label.

Here we present a protocol to test the efficacy of targeted therapies selected based on the genomic makeup of a tumor. The protocol describes identification and validation of structural DNA rearrangements, engraftment of patients&#8217; tumors into mice and testing responses to corresponding drugs.

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using bioinformatics pipelines (&#34;dry bench&#34;). Both elements are essential to produce accurate and reliable results, which are particularly critical for clinical laboratories. Targeted NGS technologies have increasingly found favor in oncology applications to help advance precision medicine objectives, yet the methods often involve disconnected and variable wet and dry bench workflows and uncoordinated reagent sets. In this report, we describe a method for sequencing challenging cancer specimens with a 21-gene panel as an example of a comprehensive targeted NGS system. The system integrates functional DNA quantification and qualification, single-tube multiplexed PCR enrichment, and library purification and normalization using analytically-verified, single-source reagents with a standalone bioinformatics suite. As a result, accurate variant calls from low-quality and low-quantity formalin-fixed, paraffin-embedded (FFPE) and fine-needle aspiration (FNA) tumor biopsies can be achieved. The method can routinely assess cancer-associated variants from an input of 400 amplifiable DNA copies, and is modular in design to accommodate new gene content. Two different types of analytically-defined controls provide quality assurance and help safeguard call accuracy with clinically-relevant samples. A flexible &#34;tag&#34; PCR step embeds platform-specific adaptors and index codes to allow sample barcoding and compatibility with common benchtop NGS instruments. Importantly, the protocol is streamlined and can produce 24 sequence-ready libraries in a single day. Finally, the approach links wet and dry bench processes by incorporating pre-analytical sample quality control results directly into the variant calling algorithms to improve mutation detection accuracy and differentiate false-negative and indeterminate calls. This targeted NGS method uses advances in both wetware and software to achieve high-depth, multiplexed sequencing and sensitive analysis of heterogeneous cancer samples for diagnostic applications.

An integrated system for targeted next-generation sequencing of oncology specimens is described. This cross-platform system is optimized for low-quality and low-quantity tumor biopsies, accommodates low DNA inputs, includes well-characterized multi-variant controls, and features a novel variant caller that is informed by quantitative pre-analytical quality control measures.

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using ...

Medicine

Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base excision repair (BER) molecular beacons. The substrate (beacon) is comprised of a deoxyoligonucleotide containing a single base lesion with a 6-Carboxyfluorescein (6-FAM) moiety conjugated to the 5'end and a Dabcyl moiety conjugated to the 3' end of the oligonucleotide. The BER molecular beacon is 43 bases in length and the sequence is designed to promote the formation of a stem-loop structure with 13 nucleotides in the loop and 15 base pairs in the stem1,2. When folded in this configuration the 6-FAM moiety is quenched by Dabcyl in a non-fluorescent manner via F&ouml;rster Resonance Energy Transfer (FRET)3,4. The lesion is positioned such that following base lesion removal and strand scission the remaining 5 base oligonucleotide containing the 6-FAM moiety is released from the stem. Release and detachment from the quencher (Dabcyl) results in an increase of fluorescence that is proportionate to the level of DNA repair. By collecting multiple reads of the fluorescence values, real-time assessment of BER activity is possible. The use of standard quantitative real-time PCR instruments allows the simultaneous analysis of numerous samples. The design of these BER molecular beacons, with a single base lesion, is amenable to kinetic analyses, BER quantification and inhibitor validation and is adaptable for quantification of DNA Repair activity in tissue and tumor cell lysates or with purified proteins. The analysis of BER activity in tumor lysates or tissue aspirates using these molecular beacons may be applicable to functional biomarker measurements. Further, the analysis of BER activity with purified proteins using this quantitative assay provides a rapid, high-throughput method for the discovery and validation of BER inhibitors.

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates. The assay yields rates of DNA Repair activity amenable to kinetic analysis and is adaptable for quantification of DNA Repair activity in tissue and tumor lysates or with purified proteins.

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base ...

Biology

Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

Efforts to detect and investigate key oncogenic mutations have proven valuable to facilitate the appropriate treatment for cancer patients. The establishment of high-throughput, massively parallel "next-generation" sequencing has aided the discovery of many such mutations. To enhance the clinical and translational utility of this technology, platforms must be high-throughput, cost-effective, and compatible with formalin-fixed paraffin embedded (FFPE) tissue samples that may yield small amounts of degraded or damaged DNA. Here, we describe the preparation of barcoded and multiplexed DNA libraries followed by hybridization-based capture of targeted exons for the detection of cancer-associated mutations in fresh frozen and FFPE tumors by massively parallel sequencing. This method enables the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus conferring high sensitivity for detecting low frequency mutations.

We describe the preparation of barcoded DNA libraries and subsequent hybridization-based exon capture for detection of key cancer-associated mutations in clinical tumor specimens by massively parallel "next generation" sequencing. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus yielding high sensitivity for detecting low frequency mutations.

Efforts  to detect and investigate key oncogenic mutations have proven valuable to  facilitate the appropriate treatment for cancer patients. The ...

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3&#39;-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

gDNA enrichment for NGS sequencing is an easy and powerful tool for the study of constitutional mutations. In this article, we present the procedure to analyse simply the complete sequence of 11 genes involved in DNA damage repair.

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

Comparative Lesions Analysis Through a Targeted Sequencing Approach

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