Characterization of Novel ctDNA Reference Materials Developed using the Genom...Thermo Fisher Scientific
Liquid biopsy diagnostic technologies have revolutionized cancer testing and therapeutic monitoring. Non-invasive sample collection removes the need for invasive and dangerous biopsies to diagnose cancer and monitor therapeutic efficacy. As liquid biopsy technologies become more sensitive, screening for early detection of cancer DNA using a blood test could become routine clinical practice. However, such technologies cannot be developed without high quality reference materials. In this study, ctDNA reference materials using the NIST Genome in a Bottle GM24385 cell line DNA were developed in a human plasma-EDTA matrix. The allelic frequency (AF), size and stability of the materials were analyzed.
Evaluation of ctDNA extraction methods and amplifiable copy number yield usin...Thermo Fisher Scientific
The use of cell-free circulating tumor DNA (ctDNA) for non-invasive cancer testing has the potential to revolutionize the field. However, emergence of an increasing number of extraction methods and detection assays is rendering laboratory workflow development much more complex and cumbersome. The use of standardized, well characterized ctDNA control materials in human plasma could facilitate the evaluation of extraction efficiency and assay performance across platforms. In this study, we use a full process ctDNA quality control material in true human plasma to demonstrate the variability of extraction yield between different ctDNA extraction kits. We also examine the correlation between the amplifiable
copy number and DNA concentration post-extraction.
Improvement of TMB Measurement by removal of Deaminated Bases in FFPE DNAThermo Fisher Scientific
Tumor mutational burden (TMB) is a positive predictive factor for response to immune-checkpoint inhibitors in certain types of cancer. The Oncomine™ Tumor Mutation Load Assay, a targeted next generation sequencing (NGS) assay, measures TMB (from 1.2Mb of coding region) and detects mutations in 409 cancer genes. The TMB values obtained using targeted sequencing are highly correlated with TMB measured by whole exome sequencing. FFPE preservation methods can lead to significant cytosine deamination of the isolated DNA, resulting in decreased sequencing quality. In these samples, uracils are propagated as thymines and result in false C>T substitutions. Analysis of the Oncomine™ TML Assay using Torrent Suite and Ion Reporter ™ software uniquely estimates the degree of deamination in fixed tissues by measuring C:G>T:A variants. This deamination score is used to assess quality of DNA extracted from FFPE tumor tissue. To minimize the influence
that excess deamination has on TMB results, we have incorporated a repair treatment to eliminate damaged targets and improve usable TMB values of DNA from damaged FFPE tumor tissue using Uracil-DNA glycosylase (UDG). The
Oncomine™ TML Assay for TMB on the Ion Gene Studio™ S5 systems in conjunction with a deamination score is informative and potentially predictive for the use of checkpoint inhibitors in multiple cancer types.
A Next-Generation Sequencing Assay to Estimate Tumor Mutation Load at > 5% Al...Thermo Fisher Scientific
Immunotherapies have shown anti-cancer effects in melanoma, NSCLC, and bladder cancer. High tumor mutation load is associated with positive responses from immune checkpoint inhibitors. However, current methods to estimate tumor mutation load often have high infrastructure needs, and require large amounts of DNA.
Hot-start DNA polymerases are commonly used in PCR for genotyping, sequencing, molecular diagnostics, and high-throughput applications. In this presentation, PCR performance of Invitrogen™ Platinum II Taq Hot-Start DNA Polymerase and Invitrogen™ AccuPrime Taq DNA Polymerase is compared in the following areas:
• PCR run time for targets of different lengths
• Amplification of AT-rich and GC-rich sequences
• Tolerance to PCR inhibitors
• Sensitivity in target detection
• Universal protocol for PCR targets of different lengths
• Multiplex PCR of 15 targets
• Product format for direct gel loading
Request a sample of Platinum II Taq enzyme at http://bit.ly/2M4U9cw
Find other PCR enzymes at http://bit.ly/2JIPrzj
Learn more about PCR at http://bit.ly/2y2aSVo
#PCR #PCREducation #Invitrogen #InvitrogenSchoolofMolBio
Analytical Validation of the Oncomine™ Comprehensive Assay v3 with FFPE and C...Thermo Fisher Scientific
Presented here is an analytical validation of OCAv3 at the Life Technologies Clinical Services Laboratory (LTCSL), a CAP-accredited and CLIA-certified clinical laboratory. Analytical validations provide evidence of consistently accurate and relevant sequencing results.
Characterization of Novel ctDNA Reference Materials Developed using the Genom...Thermo Fisher Scientific
Liquid biopsy diagnostic technologies have revolutionized cancer testing and therapeutic monitoring. Non-invasive sample collection removes the need for invasive and dangerous biopsies to diagnose cancer and monitor therapeutic efficacy. As liquid biopsy technologies become more sensitive, screening for early detection of cancer DNA using a blood test could become routine clinical practice. However, such technologies cannot be developed without high quality reference materials. In this study, ctDNA reference materials using the NIST Genome in a Bottle GM24385 cell line DNA were developed in a human plasma-EDTA matrix. The allelic frequency (AF), size and stability of the materials were analyzed.
Evaluation of ctDNA extraction methods and amplifiable copy number yield usin...Thermo Fisher Scientific
The use of cell-free circulating tumor DNA (ctDNA) for non-invasive cancer testing has the potential to revolutionize the field. However, emergence of an increasing number of extraction methods and detection assays is rendering laboratory workflow development much more complex and cumbersome. The use of standardized, well characterized ctDNA control materials in human plasma could facilitate the evaluation of extraction efficiency and assay performance across platforms. In this study, we use a full process ctDNA quality control material in true human plasma to demonstrate the variability of extraction yield between different ctDNA extraction kits. We also examine the correlation between the amplifiable
copy number and DNA concentration post-extraction.
Improvement of TMB Measurement by removal of Deaminated Bases in FFPE DNAThermo Fisher Scientific
Tumor mutational burden (TMB) is a positive predictive factor for response to immune-checkpoint inhibitors in certain types of cancer. The Oncomine™ Tumor Mutation Load Assay, a targeted next generation sequencing (NGS) assay, measures TMB (from 1.2Mb of coding region) and detects mutations in 409 cancer genes. The TMB values obtained using targeted sequencing are highly correlated with TMB measured by whole exome sequencing. FFPE preservation methods can lead to significant cytosine deamination of the isolated DNA, resulting in decreased sequencing quality. In these samples, uracils are propagated as thymines and result in false C>T substitutions. Analysis of the Oncomine™ TML Assay using Torrent Suite and Ion Reporter ™ software uniquely estimates the degree of deamination in fixed tissues by measuring C:G>T:A variants. This deamination score is used to assess quality of DNA extracted from FFPE tumor tissue. To minimize the influence
that excess deamination has on TMB results, we have incorporated a repair treatment to eliminate damaged targets and improve usable TMB values of DNA from damaged FFPE tumor tissue using Uracil-DNA glycosylase (UDG). The
Oncomine™ TML Assay for TMB on the Ion Gene Studio™ S5 systems in conjunction with a deamination score is informative and potentially predictive for the use of checkpoint inhibitors in multiple cancer types.
A Next-Generation Sequencing Assay to Estimate Tumor Mutation Load at > 5% Al...Thermo Fisher Scientific
Immunotherapies have shown anti-cancer effects in melanoma, NSCLC, and bladder cancer. High tumor mutation load is associated with positive responses from immune checkpoint inhibitors. However, current methods to estimate tumor mutation load often have high infrastructure needs, and require large amounts of DNA.
Hot-start DNA polymerases are commonly used in PCR for genotyping, sequencing, molecular diagnostics, and high-throughput applications. In this presentation, PCR performance of Invitrogen™ Platinum II Taq Hot-Start DNA Polymerase and Invitrogen™ AccuPrime Taq DNA Polymerase is compared in the following areas:
• PCR run time for targets of different lengths
• Amplification of AT-rich and GC-rich sequences
• Tolerance to PCR inhibitors
• Sensitivity in target detection
• Universal protocol for PCR targets of different lengths
• Multiplex PCR of 15 targets
• Product format for direct gel loading
Request a sample of Platinum II Taq enzyme at http://bit.ly/2M4U9cw
Find other PCR enzymes at http://bit.ly/2JIPrzj
Learn more about PCR at http://bit.ly/2y2aSVo
#PCR #PCREducation #Invitrogen #InvitrogenSchoolofMolBio
Analytical Validation of the Oncomine™ Comprehensive Assay v3 with FFPE and C...Thermo Fisher Scientific
Presented here is an analytical validation of OCAv3 at the Life Technologies Clinical Services Laboratory (LTCSL), a CAP-accredited and CLIA-certified clinical laboratory. Analytical validations provide evidence of consistently accurate and relevant sequencing results.
Creating custom gene panels for next-generation sequencing: optimization of 5...Thermo Fisher Scientific
Next-generation sequencing gene panels enable the examination of multiple genes, identifying previously described variants and discovering novel variants, to elucidate genetic disease. The challenges are substantial, including: identification of all genes of interest; assay optimization to create robust, reproducible, multiplex panels; and developing accurate, comprehensive, reproducible analysis pipelines.
Detecting minor genetic variants has become essential to cancer
and infectious disease management. Many have turned to next
generation sequencing to fill this need given the common
perception that the limit of detection (LOD) for Sanger sequencing
is somewhere between 15% to 25%1,2,3. We have discovered a
software algorithmic solution to reduce this detection limit to 5%
and have demonstrated detection at even lower allele frequencies.
Standard Sanger sequencing protocols can be used and the
method can generate the familiar electropherogram data display
with noise substantially reduced. This opens up an alternative for
detecting low level somatic variants.
The key observation that enabled this development is that the noise
underlying Sanger sequencing fluorescence data (traces) appears
to be highly correlated to the primary sequence in the data. Figure
1 shows the electropherograms from two different samples: the
control sample has the same primary sequence as the test sample
which contains a few minor variants.
A next Generation Sequencing Approach to Detect Large Rearrangements in BRCA1...Thermo Fisher Scientific
We have developed an amplicon-based NGS approach for FFPE
samples that can detect SNVs, small mutations and LRs
simultaneously. We have implemented a comprehensive
bioinformatics algorithm that detects LRs at high sensitivity, even in
the absence of control sample(s). This significantly reduces the cost
and labor for BRCA1/2 genetic analyses.
Decades of cancer research including comprehensive molecular profiling combined with the
development of a broad array of targeted therapies have created the opportunity to transform
cancer care in the near future by implementing precision oncology based approaches. An
important element of this system is the widespread availability of robust and cost-effective
multivariate profiling methods in order to characterize relevant cancer associated molecular
alterations.
Current commercially available multivariate profiling methods vary dramatically with regard to
the number of cancer genes interrogated. Given that many large scale and detailed molecular
profiling studies have been completed, the landscape of somatic alterations in solid tumors is
reasonably well-known. Furthermore, the specific gene variants that are relevant to application
of targeted therapies are also a matter of record. Therefore, we set out to define the number of
relevant cancer genes for precision oncology research based on the currently available
empirical evidence.
Tumor Mutational Load assessment of FFPE samples using an NGS based assayThermo Fisher Scientific
Understanding the molecular determinants of response to immune checkpoint blockade inhibitors is a critical unmet need for translational oncology research. Research tools to characterize the mutational landscape of cancers may potentially help identify predictive biomarkers for immuno-therapy that can be tested in future studies. Herein, we describe a targeted Ion AmpliSeq assay to determine the mutational load and signature of cancer research samples.
Computational Methods for detection of somatic mutations at 0.1% frequency fr...Thermo Fisher Scientific
Blood screening to track tumor recurrence and
resistance may improve treatment selection and
monitoring. Virtually all tumors carry somatic DNA
mutations, serving as biomarker in blood. Circulating
cell-free DNA (cfDNA) is one source of tumor DNA in
blood. Tumor DNA comes from different tumor
clones, and its abundance in plasma can be very low
at critical stages such as early recurrence or
development of resistance. This enables interest in
detecting mutation biomarkers at very low frequency
from cfDNA. We present a research use only
analysis workflow for detection of low frequency DNA
variants. Our variant calling method enables
sensitive and specific detection of somatic mutations
to 0.1% frequency.
Orthogonal Verification of Oncomine cfDNA Data with Digital PCR Using TaqMan ...Thermo Fisher Scientific
The discovery of circulating tumor DNA (ctDNA) in blood, urine
and other bodily fluids has led to a new type of non-invasive
method of characterizing cancer-causing mutations, the liquid
biopsy. With NGS technologies becoming increasingly
sensitive, down to a Limit of Detection (LOD) of 0.1%, they are
rapidly gaining traction as a valid assay for cancer genotyping
and have potential to direct cancer treatment plans. The wideangle
view provided by NGS panels, combined with digital
PCR’s zoomed-in precision detection of DNA provide a
comprehensive picture of a cancer’s genetic makeup. By
applying these complementary techniques at the appropriate
time based on the disease type and stage, cancer treatment
becomes quicker, more precise and more cost-effective in the
future. NGS and digital PCR (dPCR) together provide a
complete picture of the cancer genome.
Detection of rare mutations in tumor tissue and cell free DNA (cfDNA) allows for monitoring of tumor progression and regression for research purposes. cfDNA isolated from plasma combined with a sensitive detection method like digital PCR is non- invasive and enables earlier detection compared to conventional imaging techniques. Building on the TaqMan based Rare Mutation assay set for detection of rare mutations using digital PCR on the QuantStudio 3D Digital PCR System, we are now developing multiplex assays for simultaneous detection of several mutations. We selected relevant mutations in the EGFR and KRAS genes for our initial multiplex application: EGFR G719, EGFR exon 19 deletions, and
KRAS G12/G13. These mutations may have implications for potential future targeted therapy. Primers and probes of singleplex Rare Mutation Assays were reformulated to generate multiplex assays detecting the EGFR and KRAS mutations. All multiplex assays were tested on template composed of wild-type genomic DNA background mixed with mutant plasmid reflecting each of the mutations detected by the multiplex
assays. Initial experimental results were successful and showed excellent signal intensity and clear cluster separation when analyzed with the QuantStudio 3D AnalysisSuiteTM Cloud Software. The EGFR G719 mutations (COSM6239, COSM6253, COSM6252) were detected using a 3plex assay, EGFR exon 19 deletions (COSM12383, COSM12422, COSM12678, COSM6223, COSM6254, COSM6255) were detected using a 6plex assay, and KRAS G12/G13 mutations (COSM516,
COSM517, COSM518, COSM520, COSM521, COSM522, COSM527, COSM532) were detected using an 8plex. Multiplexing assays for three relevant mutation loci proved feasible and presents an efficient way to assess the presence and the percentage of mutations at these loci.
Rare Mutation Analysis Using Digital PCR on QuantStudio™ 3D to Verify Ion Amp...Thermo Fisher Scientific
We identified mutations in eleven cell free
(cf) DNA samples by next generation
sequencing (NGS) using the Ion AmpliSeq™
Colon & Lung Cancer Research Panel and
the Ion PGM™ System. Since detection of
low frequency mutant alleles may not always
be called confidently in NGS, we verified
results by rare mutation analysis using
digital PCR on the QuantStudio™ 3D Digital
PCR System as an independent method.
We show that frequencies detected are
consistent for both methods for low
frequency mutant alleles at and below 1%.
Comparison of Type and Time of Fixation on Tissue DNA Sequencing ResultsThermo Fisher Scientific
The effects of type and duration of tissue fixation were studied using three different
lung (LCa) cancer research samples. Each tissue sample was fixed in five different
fixatives, for three different time points in each fixative. Next generation sequencing
(NGS), tissue morphology analysis (H+E), and antigenicity (IHC) were performed
for each of the resulting samples. The analysis indicates that both time and type of
fixation impact NGS results.
Low Level Somatic Variant Detection by Sanger Sequencing of FFPE Samples for ...Thermo Fisher Scientific
DNA sequence variants play an important role in the initiation and progression of many different cancer types. The detection of germline variants at a fixed ratio by gold-standard Sanger sequencing has been well established; however, the detection of somatic mutations, especially in heterogeneous tumor samples where variants may be present at a lower level, has been more challenging. Minor Variant Finder Software (MVF) enables calling of low frequency variants at a detection level as low as 5% using Sanger sequencing.
We have developed gene-specific Sanger sequencing panels covering the entire coding region (all exons) of specific genes (e.g., TP53, KRAS, and NRAS) implicated in tumorigenesis. We initially determined variants of TP53 and KRAS from lung tumor FFPE samples by NGS using the Ion PGM™ System. We confirmed the identity and minor allele frequency of these variants by gene-specific Sanger sequencing panels analyzed by MVF.
To demonstrate the robustness and flexibility of using Sanger sequencing for oncology research, we also included variants across many different solid tumor types in a pan-cancer panel. We tested this workflow with lower amounts of DNA input (10ng, 3ng, 1ng, 0.1ng). Additionally, we have built an extended RAS panel including eight amplicons covering the most important codons (12-13, 59-61, 117 and 146) of KRAS and NRAS genes. The entire workflow and data analysis using MVF was validated on thirty-five FFPE samples derived from colon cancer biopsies by OmniSeq LLC, Buffalo, NY.
Noninvasive detection of rare mutations in blood could allow tumor monitoring for
research purposes. Research studies have suggested that cfDNA contains DNA from
tumor cells with somatic mutations that could inform on tumor progression and
therapeutic resistance. Here, we demonstrate a complete workflow from a single tube
of blood through data analysis for research samples down to a 0.1% allelic frequency.
The low abundance tumor mutations found in cfDNA requires sensitive and accurate
mutation detection. We have developed two panels that utilize an amplificationbased
assay that generates tagged DNA copies, which allows detection of low
abundance tumor mutations found in cfDNA. The two panels allow multiplex
interrogation of primary driver and resistance mutations specific to ctDNA from breast
and colon cancer. The Oncomine™ Colon cfDNA panel targets 236 hotspots within
14 genes while the Oncomine Breast cfDNA panel covers 157 hotspot mutations in
10 genes. This workflow was validated from matched single blood tubes, Streck and
K2EDTA. Additionally, the utility for cancer research was demonstrated with
concordance studies using matched FFPE and plasma from oncology samples.
Treating cancer effectively requires an understanding of the molecular alterations driving each patient’s tumor. Targeted sequencing efforts that characterize prevalent somatic alterations and require limited sample input may provide an effective diagnostic approach. Herein, we describe the design and characterization of the Oncomine™ Cancer Research Panel (OCP) that includes recurrent somatic alterations in solid tumors derived from the Oncomine™ cancer database. Using Ion AmpliSeq™ technology, we designed a DNA panel that includes assays for 73 oncogenes with 1,826 recurrent hotspot mutations, 26 tumor suppressor genes enriched for deleterious mutations, as well as 75 genes subject to recurrent focal copy gain or loss. A complementary RNA panel includes 183 assays for relevant gene fusions involving 22 fusion driver genes. Recommended sample inputs were 10 ng of nucleic acid per pool. Sequencing libraries were analyzed on an Ion Torrent™ Personal Genome Machine™. Initial testing revealed an average read depth of > 1,500X with > 95% uniformity and on target frequency. The panel was shown to reliably detect known hotspots, insertions/deletions, gene copy changes, and gene fusions in molecular standards, cell lines and formalin-fixed paraffin embedded samples. Retrospective analysis of large sample cohorts has been completed and the results of analysis of 100 lung cancer and 100 prostate cancer cases will be summarized. In addition, a prospective cohort of 100 samples from the University of Michigan Molecular Diagnostics laboratory was profiled with OCP. Overall, we achieved >95% sensitivity and specificity for detection of KRAS, EGFR and BRAF mutations and ALK gene fusions.
Quantification of Donor/Recipient Chimerism in Leukemia Samples by Digital PCRThermo Fisher Scientific
During leukemia treatment mixed chimerism occurs in which both recipient and donor cells are present in the bone marrow or peripheral blood after transplantation.
Chimerism analysis is performed to monitor peripheral blood or bone marrow in the recipient after allogenic stem cell transplantation to monitor for leukemic relapse. Observation of increasing mixed chimerism after transplantation is associated with a higher risk of relapse in acute leukemia. Previously, a quantitative PCR (qPCR) technique, using INDEL polymorphisms, was found to predict relapse in 88.2% vs. 44.4% of individuals analyzed by VNTR markers with a median anticipation period of 58 days and a sensitivity of 0.01% vs. 3%. Here we present results from research experiments performed to determine if a digital PCR (dPCR) method is able to predict relapse earlier and with greater accuracy than the qPCR method using retrospective leukemia samples. Research results showed that dPCR using data generated by the QuantStudio™ 3D Digital PCR System and the qPCR method yielded similar percent recipient chimerism values when recipient DNA was present above the 1% level. Furthermore, dPCR using the system was found to be more sensitive than the qPCR method based on the ability to detect the recipient DNA in a relapsed individual about 2 months earlier where the percent recipient chimerism was 0.2% or less. The false positive rate was close to the complete chimerism value of 0.01% for peripheral blood samples.
Ion Torrent™ Next Generation Sequencing – Detect 0.1% Low Frequency Somatic V...Thermo Fisher Scientific
Accurate detection of low-frequency somatic mutations as well as low level structural variants such as copy number variation (CNV) in circulating cell-free DNA (cfDNA) using blood samples from subjects previously diagnosed with cancer provides a potential non-invasive approach to monitor cancer status and evaluate cancer evolution in the future. We have previously reported the Oncomine™ Breast cfDNA Assay enables detection of somatic mutations in plasma down to a level of 0.1% variant allelic frequency in breast cancer relevant genes. Here we extend this technology to simultaneously detect single nucleotide variants (SNVs) as well as copy number variation (CNV) from a single cfDNA sample.
Creating custom gene panels for next-generation sequencing: optimization of 5...Thermo Fisher Scientific
Next-generation sequencing gene panels enable the examination of multiple genes, identifying previously described variants and discovering novel variants, to elucidate genetic disease. The challenges are substantial, including: identification of all genes of interest; assay optimization to create robust, reproducible, multiplex panels; and developing accurate, comprehensive, reproducible analysis pipelines.
Detecting minor genetic variants has become essential to cancer
and infectious disease management. Many have turned to next
generation sequencing to fill this need given the common
perception that the limit of detection (LOD) for Sanger sequencing
is somewhere between 15% to 25%1,2,3. We have discovered a
software algorithmic solution to reduce this detection limit to 5%
and have demonstrated detection at even lower allele frequencies.
Standard Sanger sequencing protocols can be used and the
method can generate the familiar electropherogram data display
with noise substantially reduced. This opens up an alternative for
detecting low level somatic variants.
The key observation that enabled this development is that the noise
underlying Sanger sequencing fluorescence data (traces) appears
to be highly correlated to the primary sequence in the data. Figure
1 shows the electropherograms from two different samples: the
control sample has the same primary sequence as the test sample
which contains a few minor variants.
A next Generation Sequencing Approach to Detect Large Rearrangements in BRCA1...Thermo Fisher Scientific
We have developed an amplicon-based NGS approach for FFPE
samples that can detect SNVs, small mutations and LRs
simultaneously. We have implemented a comprehensive
bioinformatics algorithm that detects LRs at high sensitivity, even in
the absence of control sample(s). This significantly reduces the cost
and labor for BRCA1/2 genetic analyses.
Decades of cancer research including comprehensive molecular profiling combined with the
development of a broad array of targeted therapies have created the opportunity to transform
cancer care in the near future by implementing precision oncology based approaches. An
important element of this system is the widespread availability of robust and cost-effective
multivariate profiling methods in order to characterize relevant cancer associated molecular
alterations.
Current commercially available multivariate profiling methods vary dramatically with regard to
the number of cancer genes interrogated. Given that many large scale and detailed molecular
profiling studies have been completed, the landscape of somatic alterations in solid tumors is
reasonably well-known. Furthermore, the specific gene variants that are relevant to application
of targeted therapies are also a matter of record. Therefore, we set out to define the number of
relevant cancer genes for precision oncology research based on the currently available
empirical evidence.
Tumor Mutational Load assessment of FFPE samples using an NGS based assayThermo Fisher Scientific
Understanding the molecular determinants of response to immune checkpoint blockade inhibitors is a critical unmet need for translational oncology research. Research tools to characterize the mutational landscape of cancers may potentially help identify predictive biomarkers for immuno-therapy that can be tested in future studies. Herein, we describe a targeted Ion AmpliSeq assay to determine the mutational load and signature of cancer research samples.
Computational Methods for detection of somatic mutations at 0.1% frequency fr...Thermo Fisher Scientific
Blood screening to track tumor recurrence and
resistance may improve treatment selection and
monitoring. Virtually all tumors carry somatic DNA
mutations, serving as biomarker in blood. Circulating
cell-free DNA (cfDNA) is one source of tumor DNA in
blood. Tumor DNA comes from different tumor
clones, and its abundance in plasma can be very low
at critical stages such as early recurrence or
development of resistance. This enables interest in
detecting mutation biomarkers at very low frequency
from cfDNA. We present a research use only
analysis workflow for detection of low frequency DNA
variants. Our variant calling method enables
sensitive and specific detection of somatic mutations
to 0.1% frequency.
Orthogonal Verification of Oncomine cfDNA Data with Digital PCR Using TaqMan ...Thermo Fisher Scientific
The discovery of circulating tumor DNA (ctDNA) in blood, urine
and other bodily fluids has led to a new type of non-invasive
method of characterizing cancer-causing mutations, the liquid
biopsy. With NGS technologies becoming increasingly
sensitive, down to a Limit of Detection (LOD) of 0.1%, they are
rapidly gaining traction as a valid assay for cancer genotyping
and have potential to direct cancer treatment plans. The wideangle
view provided by NGS panels, combined with digital
PCR’s zoomed-in precision detection of DNA provide a
comprehensive picture of a cancer’s genetic makeup. By
applying these complementary techniques at the appropriate
time based on the disease type and stage, cancer treatment
becomes quicker, more precise and more cost-effective in the
future. NGS and digital PCR (dPCR) together provide a
complete picture of the cancer genome.
Detection of rare mutations in tumor tissue and cell free DNA (cfDNA) allows for monitoring of tumor progression and regression for research purposes. cfDNA isolated from plasma combined with a sensitive detection method like digital PCR is non- invasive and enables earlier detection compared to conventional imaging techniques. Building on the TaqMan based Rare Mutation assay set for detection of rare mutations using digital PCR on the QuantStudio 3D Digital PCR System, we are now developing multiplex assays for simultaneous detection of several mutations. We selected relevant mutations in the EGFR and KRAS genes for our initial multiplex application: EGFR G719, EGFR exon 19 deletions, and
KRAS G12/G13. These mutations may have implications for potential future targeted therapy. Primers and probes of singleplex Rare Mutation Assays were reformulated to generate multiplex assays detecting the EGFR and KRAS mutations. All multiplex assays were tested on template composed of wild-type genomic DNA background mixed with mutant plasmid reflecting each of the mutations detected by the multiplex
assays. Initial experimental results were successful and showed excellent signal intensity and clear cluster separation when analyzed with the QuantStudio 3D AnalysisSuiteTM Cloud Software. The EGFR G719 mutations (COSM6239, COSM6253, COSM6252) were detected using a 3plex assay, EGFR exon 19 deletions (COSM12383, COSM12422, COSM12678, COSM6223, COSM6254, COSM6255) were detected using a 6plex assay, and KRAS G12/G13 mutations (COSM516,
COSM517, COSM518, COSM520, COSM521, COSM522, COSM527, COSM532) were detected using an 8plex. Multiplexing assays for three relevant mutation loci proved feasible and presents an efficient way to assess the presence and the percentage of mutations at these loci.
Rare Mutation Analysis Using Digital PCR on QuantStudio™ 3D to Verify Ion Amp...Thermo Fisher Scientific
We identified mutations in eleven cell free
(cf) DNA samples by next generation
sequencing (NGS) using the Ion AmpliSeq™
Colon & Lung Cancer Research Panel and
the Ion PGM™ System. Since detection of
low frequency mutant alleles may not always
be called confidently in NGS, we verified
results by rare mutation analysis using
digital PCR on the QuantStudio™ 3D Digital
PCR System as an independent method.
We show that frequencies detected are
consistent for both methods for low
frequency mutant alleles at and below 1%.
Comparison of Type and Time of Fixation on Tissue DNA Sequencing ResultsThermo Fisher Scientific
The effects of type and duration of tissue fixation were studied using three different
lung (LCa) cancer research samples. Each tissue sample was fixed in five different
fixatives, for three different time points in each fixative. Next generation sequencing
(NGS), tissue morphology analysis (H+E), and antigenicity (IHC) were performed
for each of the resulting samples. The analysis indicates that both time and type of
fixation impact NGS results.
Low Level Somatic Variant Detection by Sanger Sequencing of FFPE Samples for ...Thermo Fisher Scientific
DNA sequence variants play an important role in the initiation and progression of many different cancer types. The detection of germline variants at a fixed ratio by gold-standard Sanger sequencing has been well established; however, the detection of somatic mutations, especially in heterogeneous tumor samples where variants may be present at a lower level, has been more challenging. Minor Variant Finder Software (MVF) enables calling of low frequency variants at a detection level as low as 5% using Sanger sequencing.
We have developed gene-specific Sanger sequencing panels covering the entire coding region (all exons) of specific genes (e.g., TP53, KRAS, and NRAS) implicated in tumorigenesis. We initially determined variants of TP53 and KRAS from lung tumor FFPE samples by NGS using the Ion PGM™ System. We confirmed the identity and minor allele frequency of these variants by gene-specific Sanger sequencing panels analyzed by MVF.
To demonstrate the robustness and flexibility of using Sanger sequencing for oncology research, we also included variants across many different solid tumor types in a pan-cancer panel. We tested this workflow with lower amounts of DNA input (10ng, 3ng, 1ng, 0.1ng). Additionally, we have built an extended RAS panel including eight amplicons covering the most important codons (12-13, 59-61, 117 and 146) of KRAS and NRAS genes. The entire workflow and data analysis using MVF was validated on thirty-five FFPE samples derived from colon cancer biopsies by OmniSeq LLC, Buffalo, NY.
Noninvasive detection of rare mutations in blood could allow tumor monitoring for
research purposes. Research studies have suggested that cfDNA contains DNA from
tumor cells with somatic mutations that could inform on tumor progression and
therapeutic resistance. Here, we demonstrate a complete workflow from a single tube
of blood through data analysis for research samples down to a 0.1% allelic frequency.
The low abundance tumor mutations found in cfDNA requires sensitive and accurate
mutation detection. We have developed two panels that utilize an amplificationbased
assay that generates tagged DNA copies, which allows detection of low
abundance tumor mutations found in cfDNA. The two panels allow multiplex
interrogation of primary driver and resistance mutations specific to ctDNA from breast
and colon cancer. The Oncomine™ Colon cfDNA panel targets 236 hotspots within
14 genes while the Oncomine Breast cfDNA panel covers 157 hotspot mutations in
10 genes. This workflow was validated from matched single blood tubes, Streck and
K2EDTA. Additionally, the utility for cancer research was demonstrated with
concordance studies using matched FFPE and plasma from oncology samples.
Treating cancer effectively requires an understanding of the molecular alterations driving each patient’s tumor. Targeted sequencing efforts that characterize prevalent somatic alterations and require limited sample input may provide an effective diagnostic approach. Herein, we describe the design and characterization of the Oncomine™ Cancer Research Panel (OCP) that includes recurrent somatic alterations in solid tumors derived from the Oncomine™ cancer database. Using Ion AmpliSeq™ technology, we designed a DNA panel that includes assays for 73 oncogenes with 1,826 recurrent hotspot mutations, 26 tumor suppressor genes enriched for deleterious mutations, as well as 75 genes subject to recurrent focal copy gain or loss. A complementary RNA panel includes 183 assays for relevant gene fusions involving 22 fusion driver genes. Recommended sample inputs were 10 ng of nucleic acid per pool. Sequencing libraries were analyzed on an Ion Torrent™ Personal Genome Machine™. Initial testing revealed an average read depth of > 1,500X with > 95% uniformity and on target frequency. The panel was shown to reliably detect known hotspots, insertions/deletions, gene copy changes, and gene fusions in molecular standards, cell lines and formalin-fixed paraffin embedded samples. Retrospective analysis of large sample cohorts has been completed and the results of analysis of 100 lung cancer and 100 prostate cancer cases will be summarized. In addition, a prospective cohort of 100 samples from the University of Michigan Molecular Diagnostics laboratory was profiled with OCP. Overall, we achieved >95% sensitivity and specificity for detection of KRAS, EGFR and BRAF mutations and ALK gene fusions.
Quantification of Donor/Recipient Chimerism in Leukemia Samples by Digital PCRThermo Fisher Scientific
During leukemia treatment mixed chimerism occurs in which both recipient and donor cells are present in the bone marrow or peripheral blood after transplantation.
Chimerism analysis is performed to monitor peripheral blood or bone marrow in the recipient after allogenic stem cell transplantation to monitor for leukemic relapse. Observation of increasing mixed chimerism after transplantation is associated with a higher risk of relapse in acute leukemia. Previously, a quantitative PCR (qPCR) technique, using INDEL polymorphisms, was found to predict relapse in 88.2% vs. 44.4% of individuals analyzed by VNTR markers with a median anticipation period of 58 days and a sensitivity of 0.01% vs. 3%. Here we present results from research experiments performed to determine if a digital PCR (dPCR) method is able to predict relapse earlier and with greater accuracy than the qPCR method using retrospective leukemia samples. Research results showed that dPCR using data generated by the QuantStudio™ 3D Digital PCR System and the qPCR method yielded similar percent recipient chimerism values when recipient DNA was present above the 1% level. Furthermore, dPCR using the system was found to be more sensitive than the qPCR method based on the ability to detect the recipient DNA in a relapsed individual about 2 months earlier where the percent recipient chimerism was 0.2% or less. The false positive rate was close to the complete chimerism value of 0.01% for peripheral blood samples.
Ion Torrent™ Next Generation Sequencing – Detect 0.1% Low Frequency Somatic V...Thermo Fisher Scientific
Accurate detection of low-frequency somatic mutations as well as low level structural variants such as copy number variation (CNV) in circulating cell-free DNA (cfDNA) using blood samples from subjects previously diagnosed with cancer provides a potential non-invasive approach to monitor cancer status and evaluate cancer evolution in the future. We have previously reported the Oncomine™ Breast cfDNA Assay enables detection of somatic mutations in plasma down to a level of 0.1% variant allelic frequency in breast cancer relevant genes. Here we extend this technology to simultaneously detect single nucleotide variants (SNVs) as well as copy number variation (CNV) from a single cfDNA sample.
This talk outlines the general steps for project management in rapid development (design to data in two weeks) of a novel digital PCR assay to validate and quantify low frequency variants discovered by sequencing (NGS) of a targeted comprehensive cancer gene panel by the Ion Torrent PGM on PDX models of metastatic colon cancer and spheroid (3-D) cell cultures.
Goal: If the potential driver mutation is validated, treat both (PDX model & cell culture) with small molecule drugs, investigate coincident response.
Principle, Procedure and applications of Digital PCR.pptxVikramadityaupmanyu
Digital PCR (dPCR) is the new generation PCR that enables absolute quantification of target gene by separating reac¬tion mixture in several compartments. In this system, copies of target nucleic acid are distributed randomly from 0, 1 or many in the several small volume compartments. Amplification is occurred in the compartment and resulting absorbance is measured. Integrated fluidic circuits, chip based microchambers, and water in oil droplets are the methods are used for separation of reaction mixture in to several compartments. BioMark HD system (Fluidigm, USA) and QuantStudio3D system (Thermofisher Scientific, USA) uses integrated fluidic circuits harbor¬ing 10000 to 40000 microchambers and integrated chip containing 20000 microchambers, respectively. In droplet digital PCR System (Biorad, USA), reaction mixture is separated into 20,000 water in droplets. After cycles of reaction using any of the above technologies, fluorescence is detected with an imaging sys¬tem in the each compartment and copy numbers of the target is calculated with imaging software.
In droplets digital PCR, after amplification, droplets containing target gene are detected by fluorescence and scored as positive, and droplets without fluorescence are scored as negative. Poisson statistical analysis of the numbers of positive and negative droplets yields absolute quantitation of the target sequence. Digital PCR is the preferred technique for absolute quantification of target gene without need of standard curve and higher sensitivity and produces highly reproducible results, and also less susceptible to inhibitors than conventional RT-qPCR
Multicopy reference assay (MRef) — a superior normalizer of sample input in D...QIAGEN
Copy number variations (CNVs) and alterations (CNAs) are a source of genetic diversity in humans and are often pathogenic. Numerous CNVs and CNAs are being identified with various genome analysis platforms, including array comparative genomic hybridization (aCGH), single nucleotide polymorphism (SNP) genotyping platforms, and next-generation sequencing. Independent verification of copy number changes is a critical step. Quantitative real-time PCR (qPCR) is a classic method to verify microarray copy number findings. Traditional copy number assays that use qPCR typically rely on a putative single-copy gene reference assay (e.g., RNase P or TERT) to normalize the DNA input for downstream ΔΔCT-based copy number calculation for comparison to a reference genome. When applied to cancer samples, these single-copy reference assays may no longer be a reliable indicator of DNA input due to the presence of complex chromosome composition (both in chromosome number and structure). To meet the need for an accurate DNA input normalizer, especially for heterogeneous tumor samples, QIAGEN developed a multicopy reference (MRef) assay for real-time PCR copy number analysis. This assay, in conjunction with QIAGEN’s greater than 10 million genomewide copy number assays and pathway- and disease-focused copy number PCR arrays (Figure 1), provides a successful solution for copy number analysis. This article will address the assay design considerations, development, and performance of this multicopy reference (MRef) assay.
Rapid and accurate Cancer somatic mutation profiling with the qBiomarker Soma...QIAGEN
QIAGEN has developed real-time PCR-based qBiomarker Somatic Mutation PCR Arrays for pathway- and disease-focused mutation profiling. By combining allele-specific amplification and 5' hydrolysis probe detection, the PCR assays on these arrays detect as little as 0.01% somatic mutation in a background of wild-type genomic DNA. These assays have consistent and reliable mutation detection performance in different sample types (including fresh, frozen, or formalin-fixed samples), and with varying sample quality. In application examples, the PCR-based mutation detection results are consistent with Pyrosequencing results for the same samples. The qBiomarker Somatic Mutation PCR Arrays, combining laboratory-verified assays, comprehensive content, and integrated data analysis software, are highly suited for identifying somatic mutations in the context of biological pathways and diseases.
How to do successful gene expression analysis - Siena 20100625Biogazelle
Despite its conceptual and practical simplicity, qPCR based expression analysis involves multiple steps, all of which need to be perfect in order to obtain reliable results in the end. This presentation describes points of attention, potential pitfalls and suggestions for improvements on every step along the workflow. By implementing these guidelines in your experiments you increase the chance of doing successful gene expression analysis.
Development of Quality Control Materials for Characterization of Comprehensiv...Thermo Fisher Scientific
Targeted next-generation sequencing (NGS) panels can detect hundreds of mutations in key genes using amplification based and hybrid-capture based NGS technologies. Although NGS technology is a powerful tool, optimizing and characterizing test performance on hundreds of variants is extremely challenging, time consuming, and expensive. Samples must be sourced, variants identified and orthogonally confirmed, then quantified and diluted. This effort is then multiplied across dozens of samples, and then samples must be run over many runs and days to assess assay reproducibility, precision, sensitivity, etc. In this study, we developed a novel reference material, experimental design, and analysis pipeline that allows for highly streamlined NGS assay characterization, enabling thorough test characterization across 500+ variants within only 6 runs.
Clinical Utility of Droplet Digital PCR on Liquid Biopsies from Patients with...Kate Barlow
The Treatment Resistance Team at the Institute of Cancer Research has been using plasma to interrogate resistance in castration-resistant prostate cancer (CRPC) and develop biomarkers for selecting treatment. Using targeted next-generation sequencing and droplet digital PCR on cfDNA from sequential plasma samples AR mutations was found to emerge with resistance to abiraterone and enzalutamide. A strong association between plasma AR aberrations in the form of AR gain and mutations and resistance to abiraterone or enzalutamide in CRPC patients was also seen, supporting the clinical utility of cfDNA studies in metastatic prostate cancer.
Daniel Wetterskog, Senior Scientist, Institute of Cancer Research, UK
The OncoScan(TM) platform for analysis of copy number and somatic mutations i...Lawrence Greenfield
The OncoScan microarray offers high-quality copy number, genotype, and somatic mutation data with whole-genome coverage and high resolution in cancer genes for use with challenging FFPE samples.
1. CytoScan®
HD copy number confirmation using real-time PCR
and USB®
VeriQuest®
SYBR®
Green qPCR Master Mix
Detection of human copy number variation (CNV) is rapidly becoming more relevant in human disease assessment. The CytoScan
HD Cytogenetics Solution provides a genome-wide approach that enables high-resolution DNA copy number analysis. In this proof
of principle study conducted by Affymetrix in collaboration with a leading diagnostic laboratory, CytoScan HD copy number variants
were confirmed with 100% concordance using VeriQuest SYBR Green real-time PCR.
Advantages of VeriQuest real-time PCR
n Improved efficiency and cost effectiveness compared to traditional cytogenetics techniques such as karyotyping and fluorescent
in situ hybridization (FISH)
n Cost effective VeriQuest SYBR Green qPCR compared to TaqMan®
assays
n Proven workflow performance
n Adoption by cytogenetics community
Proof of principle study
Human DNA samples, that were identified to have copy number variations using CytoScan HD, were sent to Affymetrix and tested
using VeriQuest SYBR Green qPCR Master Mix [PN 75600]. Genomic location, the size of the aberration, and genes of interest
within the aberrations were provided by the diagnostic laboratory. For the 20 samples tested, the nature of the aberration (duplica-
tion or deletion or normal/control) was revealed for 10 samples at the start of the study, while the confirmation of the remaining 10
samples was conducted in a single-blind study.
Two real-time PCR primer sets per aberration were designed using publicly available web tools (Primer3Plus, NCBI Primer-BLAST,
UCSC In Silico-PCR) to interrogate the indicated genes of interest. These custom primers were purchased from IDT. All primer sets
were tested in a standard curve assay to determine the specificity and efficiency for detecting CNVs. Primer pair PCR efficiency was
100 ± 10% for 98% of the primer sets (5 point standard curves, 2-fold dilution series). Primer efficiencies for the genes of interest
and the reference were within 10% of each other. PCR reactions were run on agarose gels and showed the expected product length
with single bands.
Relative genomic dosage was measured for the indicated genes relative to a reference gene (usually RNase P). Figure 1 shows repre-
sentative data for a gene with a deletion and a gene with duplication. The study compared each subject to 4 normal samples, with
one of the normal samples as the calibrator for the relative genomic dosage calculation. Table 1 lists the CytoScan HD CNV data
(aberration chromosomal location, copy number) and the genes of interest selected by the diagnostic lab for each sample. It also con-
tains the theoretically expected and the real-time PCR measured relative genomic dosage, and the corresponding copy number. Copy
number variants were confirmed with 100% concordance for both the known and single-blind, unknown samples with VeriQuest
SYBR Green qPCR Master Mix.
WhitePaper
Fig 1. Examples of relative genomic dosage measurements with VeriQuest SYBR Green qPCR Master Mix
VeriQuest SYBR Green qPCR assay compares subject with aberration to normal controls. Relative genomic dosage is calculated for the gene of interest relative to
a reference gene based on the ∆∆Ct method. The relative genomic dosage of one of the normal controls (referred to as the calibrator) is set at 1. Error bars are
calculated from 3 replicates and show ± 2 standard deviations from the mean representing a 95% confidence interval.
1.00 0.98 0.96 1.00
0.51
0.0
0.5
1.0
1.5
2.0
calibrator normal normal normal subject
Relativegenomicdosage
1X deletion (CTNS)
0.0
0.5
1.0
1.5
2.0
calibrator normal normal normal subject
Relativegenomicdosage
3X duplication (ASMT)
1.00 1.00 1.03 0.99
1.47
![CytoScan®
HD copy number confirmation using real-time PCR
and USB®
VeriQuest®
SYBR®
Green qPCR Master Mix
Detection of human copy number variation (CNV) is rapidly becoming more relevant in human disease assessment. The CytoScan
HD Cytogenetics Solution provides a genome-wide approach that enables high-resolution DNA copy number analysis. In this proof
of principle study conducted by Affymetrix in collaboration with a leading diagnostic laboratory, CytoScan HD copy number variants
were confirmed with 100% concordance using VeriQuest SYBR Green real-time PCR.
Advantages of VeriQuest real-time PCR
n Improved efficiency and cost effectiveness compared to traditional cytogenetics techniques such as karyotyping and fluorescent
in situ hybridization (FISH)
n Cost effective VeriQuest SYBR Green qPCR compared to TaqMan®
assays
n Proven workflow performance
n Adoption by cytogenetics community
Proof of principle study
Human DNA samples, that were identified to have copy number variations using CytoScan HD, were sent to Affymetrix and tested
using VeriQuest SYBR Green qPCR Master Mix [PN 75600]. Genomic location, the size of the aberration, and genes of interest
within the aberrations were provided by the diagnostic laboratory. For the 20 samples tested, the nature of the aberration (duplica-
tion or deletion or normal/control) was revealed for 10 samples at the start of the study, while the confirmation of the remaining 10
samples was conducted in a single-blind study.
Two real-time PCR primer sets per aberration were designed using publicly available web tools (Primer3Plus, NCBI Primer-BLAST,
UCSC In Silico-PCR) to interrogate the indicated genes of interest. These custom primers were purchased from IDT. All primer sets
were tested in a standard curve assay to determine the specificity and efficiency for detecting CNVs. Primer pair PCR efficiency was
100 ± 10% for 98% of the primer sets (5 point standard curves, 2-fold dilution series). Primer efficiencies for the genes of interest
and the reference were within 10% of each other. PCR reactions were run on agarose gels and showed the expected product length
with single bands.
Relative genomic dosage was measured for the indicated genes relative to a reference gene (usually RNase P). Figure 1 shows repre-
sentative data for a gene with a deletion and a gene with duplication. The study compared each subject to 4 normal samples, with
one of the normal samples as the calibrator for the relative genomic dosage calculation. Table 1 lists the CytoScan HD CNV data
(aberration chromosomal location, copy number) and the genes of interest selected by the diagnostic lab for each sample. It also con-
tains the theoretically expected and the real-time PCR measured relative genomic dosage, and the corresponding copy number. Copy
number variants were confirmed with 100% concordance for both the known and single-blind, unknown samples with VeriQuest
SYBR Green qPCR Master Mix.
WhitePaper
Fig 1. Examples of relative genomic dosage measurements with VeriQuest SYBR Green qPCR Master Mix
VeriQuest SYBR Green qPCR assay compares subject with aberration to normal controls. Relative genomic dosage is calculated for the gene of interest relative to
a reference gene based on the ∆∆Ct method. The relative genomic dosage of one of the normal controls (referred to as the calibrator) is set at 1. Error bars are
calculated from 3 replicates and show ± 2 standard deviations from the mean representing a 95% confidence interval.
1.00 0.98 0.96 1.00
0.51
0.0
0.5
1.0
1.5
2.0
calibrator normal normal normal subject
Relativegenomicdosage
1X deletion (CTNS)
0.0
0.5
1.0
1.5
2.0
calibrator normal normal normal subject
Relativegenomicdosage
3X duplication (ASMT)
1.00 1.00 1.03 0.99
1.47](https://image.slidesharecdn.com/42a4bdb9-8851-417d-a772-52785cc2bd79-161123140155/85/Cytoscan_Copy_Number_Confirmation_with_SYBR_Green_qPCR_white_paper-1-320.jpg)
