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The presence of circulating (cell-free) nucleic acids in the bloodstream offers a potential non-invasive approach to monitor disease status and guide treatment options. In past years, increasing interest has been shown for circulating RNA; especially circulating small RNAs for their potential application as biomarkers that may lead toward more effective diagnosis and prognosis in the future. However, widespread inconsistencies have been observed among the studies due to biases generated during sample collection, handling, RNA extraction and analysis. We have developed a complete workflow that includes blood collection, plasma preparation, circulating RNA extraction, followed by expression analysis and gene fusion detection on Ion TorrentTM Next-Generation Sequencing platforms. Blood plasma research samples from normal research samples were utilized for circulating RNA isolation following a TRIzolTM LS Reagent and mirVanaTM miRNA Isolation Kit-based method to maximize circulating RNA recovery. Ion AmpliSeqTM library preparation was performed on purified circulating RNA using either Ion AmpliSeq Transcriptome panel for expression profiling of 21K coding and non-coding genes, or an Ion AmpliSeq panel targeting fusion transcript detection from RNA. Ion AmpliSeq Transcriptome data was analyzed using ampliSeqRNA plugin in Torrent Suite™ Software. ~3000 genes were detected in cfDNA from plasma research samples with high correlation (r>0.8) observed between normal research samples. Ion Reporter™ Software was used to analyze fusion transcript panel data. Detection of fusion gene transcripts was demonstrated by spiking trace amounts of RNA from a fusion positive cell line into circulating RNA from normal research samples, indicating high sensitivity of the detection system. In summary, this study demonstrated the feasibility of gene expression profiling and gene fusion detection from circulating RNA in plasma research samples on Ion Torrent NGS platforms.Fusion Gene Detection and Gene Expression Analysis of Circulating RNA in Plas...
Fusion Gene Detection and Gene Expression Analysis of Circulating RNA in Plas...Thermo Fisher Scientific
Fusion transcripts resulting from translocation events in the oncogenic driver
genes ALK, RET, ROS1, and NTRK1 play an important role in lung
adenocarcinoma. There is a need to detect these fusion transcripts with up to
date technologies as they may serve as viable therapeutic targets. We have
utilized a targeted sequencing approach and developed an Ion AmpliSeq™ RNA
Lung Fusion panel, a workflow, and an Ion Reporter™ analysis solution to detect
these known fusion events. The panel detects transcripts from 37 ALK, 9 RET, 15
ROS1, and 11 NTRK fusion variants along with 5 housekeeping genes to serve
as internal controls. The workflow is FFPE compatible requiring an input of only
10 ng of total RNA with the capacity to multiplex up to 16 libraries on a single Ion
318™ chip. The panel was initially validated using 10ng of total RNA from a
cocktail of 3 cell lines containing known lung cancer fusions (H2228 – EML4-ALK
variant 3a and 3b, HCC78 – SLC34A2-ROS1 and LC-2/ad – CCDC6-RET). The
library was sequenced using the Ion PGM™ system and analyzed with the
AmpliSeq™ RNA Lung Fusion workflow in Ion Reporter™. Analysis showed that
the positive control sample contained all expected fusions and control genes and
reported zero false positives fusions. This multiplexed fusion transcript targeted
sequencing solution is currently being validated by all members of the
OncoNetwork Consortium who will test lung cancer tissue samples that have
been well characterized by FISH, real-time PCR, IHC, and/or massarray. Initial
results from OncoNetwork Consortia members reveal 100% concordance
between the AmpliSeq™ RNA Lung Fusion panel and FISH in 25 lung tissue
samples.Verification of an Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel, workf...
Verification of an Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel, workf...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.A next Generation Sequencing Approach to Detect Large Rearrangements in BRCA1...
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Recommended
The presence of circulating (cell-free) nucleic acids in the bloodstream offers a potential non-invasive approach to monitor disease status and guide treatment options. In past years, increasing interest has been shown for circulating RNA; especially circulating small RNAs for their potential application as biomarkers that may lead toward more effective diagnosis and prognosis in the future. However, widespread inconsistencies have been observed among the studies due to biases generated during sample collection, handling, RNA extraction and analysis. We have developed a complete workflow that includes blood collection, plasma preparation, circulating RNA extraction, followed by expression analysis and gene fusion detection on Ion TorrentTM Next-Generation Sequencing platforms. Blood plasma research samples from normal research samples were utilized for circulating RNA isolation following a TRIzolTM LS Reagent and mirVanaTM miRNA Isolation Kit-based method to maximize circulating RNA recovery. Ion AmpliSeqTM library preparation was performed on purified circulating RNA using either Ion AmpliSeq Transcriptome panel for expression profiling of 21K coding and non-coding genes, or an Ion AmpliSeq panel targeting fusion transcript detection from RNA. Ion AmpliSeq Transcriptome data was analyzed using ampliSeqRNA plugin in Torrent Suite™ Software. ~3000 genes were detected in cfDNA from plasma research samples with high correlation (r>0.8) observed between normal research samples. Ion Reporter™ Software was used to analyze fusion transcript panel data. Detection of fusion gene transcripts was demonstrated by spiking trace amounts of RNA from a fusion positive cell line into circulating RNA from normal research samples, indicating high sensitivity of the detection system. In summary, this study demonstrated the feasibility of gene expression profiling and gene fusion detection from circulating RNA in plasma research samples on Ion Torrent NGS platforms.Fusion Gene Detection and Gene Expression Analysis of Circulating RNA in Plas...
Fusion Gene Detection and Gene Expression Analysis of Circulating RNA in Plas...Thermo Fisher Scientific
Fusion transcripts resulting from translocation events in the oncogenic driver
genes ALK, RET, ROS1, and NTRK1 play an important role in lung
adenocarcinoma. There is a need to detect these fusion transcripts with up to
date technologies as they may serve as viable therapeutic targets. We have
utilized a targeted sequencing approach and developed an Ion AmpliSeq™ RNA
Lung Fusion panel, a workflow, and an Ion Reporter™ analysis solution to detect
these known fusion events. The panel detects transcripts from 37 ALK, 9 RET, 15
ROS1, and 11 NTRK fusion variants along with 5 housekeeping genes to serve
as internal controls. The workflow is FFPE compatible requiring an input of only
10 ng of total RNA with the capacity to multiplex up to 16 libraries on a single Ion
318™ chip. The panel was initially validated using 10ng of total RNA from a
cocktail of 3 cell lines containing known lung cancer fusions (H2228 – EML4-ALK
variant 3a and 3b, HCC78 – SLC34A2-ROS1 and LC-2/ad – CCDC6-RET). The
library was sequenced using the Ion PGM™ system and analyzed with the
AmpliSeq™ RNA Lung Fusion workflow in Ion Reporter™. Analysis showed that
the positive control sample contained all expected fusions and control genes and
reported zero false positives fusions. This multiplexed fusion transcript targeted
sequencing solution is currently being validated by all members of the
OncoNetwork Consortium who will test lung cancer tissue samples that have
been well characterized by FISH, real-time PCR, IHC, and/or massarray. Initial
results from OncoNetwork Consortia members reveal 100% concordance
between the AmpliSeq™ RNA Lung Fusion panel and FISH in 25 lung tissue
samples.Verification of an Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel, workf...
Verification of an Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel, workf...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.A next Generation Sequencing Approach to Detect Large Rearrangements in BRCA1...
A next Generation Sequencing Approach to Detect Large Rearrangements in BRCA1...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.Low Level Somatic Variant Detection by Sanger Sequencing of FFPE Samples for ...
Low Level Somatic Variant Detection by Sanger Sequencing of FFPE Samples for ...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%.Rare Mutation Analysis Using Digital PCR on QuantStudio™ 3D to Verify Ion Amp...
Rare Mutation Analysis Using Digital PCR on QuantStudio™ 3D to Verify Ion Amp...Thermo Fisher Scientific
As personalized cancer care evolves, the patient’s nucleic acid becomes ever so important to provide valuable information regarding their genetic makeup and disease state. Common sample types for these analyses include biopsies, which can be very limited in material making the downstream measurement of more than one analyte rather difficult. Obtaining another biopsy, using a different section or splitting the sample can be problematic because of tumor heterogeneity. Even adjacent areas of the same tumor tissue can result in different RNA/DNA profiles so the ability to isolate multiple analytes from the same sample offer a number of benefits, which include preserving samples and data consistency eliminating any sample to sample variation. As more tests are developed to simultaneously monitor genetic alterations, there is a strong need to efficiently isolate both DNA and RNA from the same starting sample in a format compatible with high-throughput processing.
High-throughput processing to maximize genomic analysis through simultaneous ...
High-throughput processing to maximize genomic analysis through simultaneous ...Thermo Fisher Scientific
Research has shown that circulating tumor DNA (ctDNA) is
informative of tumor load and tumor evolution in both solid and
hematological cancer. The ability to detect mutations in ctDNA
holds the promise for an accurate and non-invasive approach to
assess minimum residual disease as well as treatment
response in the future. However, as ctDNA often makes up only
a small fraction of cell-free DNA recovered from the plasma,
traditional methods of targeted sequencing research often face
a poor signal-to-noise ratio that cannot be overcome with deep
coverage.
Here we present a novel research method that is capable of
detecting ultra-rare mutations at allelic frequency below 0.5%.
This approach leverages target multiplexing capabilities of the
Ion AmpliSeq™ technology with some important modifications
to the sample preparation procedures. The new protocol
requires as little as 20 ng of input DNA and offers a sample-toanswer
turn-around time in under 24 hours. To support the
analysis of this new approach, we have further developed a
novel Bayesian statistics that models the propagation of
potential artifacts introduced during amplification and sampling
effects during sequencing to differentiate false positives
(variants observed in sequencing data that were not present in
input DNA) from true mutations that were present at very low
levels in the original research sample.
We successfully applied this new method to detect spike-in
mutant DNA in both cell line (Coriell GM24385) and cfDNA
samples. Specifically, we demonstrated the detection of 140
COSMIC genomic aberrations found in 23 frequently mutated
genes. In preliminary study, the method achieved greater 90%
sensitivity and specificity.Successful detection of 40 COSMIC hotspot mutations at allelic frequency belo...
Successful detection of 40 COSMIC hotspot mutations at allelic frequency belo...Thermo Fisher Scientific
We have developed a research method to
prepare Ion AmpliSeq libraries directly from small
amounts of biological samples such as whole
blood, saliva, buccal swabs and even FFPE
sections.
• The direct library prep methods were automated
on the Ion Chef reducing user interaction to
loading reagents and samples on to the deck of
the Ion Chef.
• Sequencing metrics were comparable or
improved for the samples used directly without
purification as compared to purified DNA controls.
Additionally, as shown for buccal swab samples,
there was no detrimental effect on variant calling
results.
• Extremely small samples were successfully
processed with this method, which reduces the
total time from sample to sequence to 24 hours.Preparing libraries directly from archived FFPE sections blood, saliva, and b...
Preparing libraries directly from archived FFPE sections blood, saliva, and b...Thermo Fisher Scientific
The presence of certain chromosomal Header
rearrangements and the subsequent fusion
gene derived from translocations has been
implicated in a number of cancers. Hundreds of
translocations have been described in the
literature recently but the need to efficiently
detect and further characterize these
chromosomal translocations is growing
exponentially. The two main methods to identify
and monitor translocations, fluorescent in situ
hybridization (FISH) and comparative genomic
hybridization (CGH) are challenging, labor
intensive, the information obtained is limited,
and sensitivity is rather low. Common sample
types for these analyses are biopsies or small
tumors, which are very limited in material
making the downstream measurement of more
than one analyte rather difficult; obtaining
another biopsy, using a different section or
splitting the sample can raise issues of tumor
heterogeneity. The ability to study mutation
status as well as measuring fusion transcript
expression from the same sample is powerful
because you’re maximizing the information
obtained from a single precious sample and
eliminating any sample to sample variation.
Here we describe the efficient isolation of two
valuable analytes, RNA and DNA, from the
same starting sample without splitting, followed
by versatile and informative downstream
analysis. This methodology has been applied to
FFPE and degraded samples as well as fresh
tissues, cells and blood. DNA and RNA were
recovered from the same non-small cell lung
adenocarcinoma sample and both mutation
analysis, as well as fusion transcript detection
was performed using the Ion Torrent PGM™
platform on the same Ion 318™ chip. Using
10ng of DNA and 10ng of RNA input, we
applied the Ion AmpliSeq™ Colon and Lung
Cancer panel to analyze over 500 COSMIC
mutations in 22 genes and the Ion AmpliSeq™
RNA Lung Fusion panel to detect 40 different
fusion transcripts.Hotspot mutation and fusion transcript detection from the same non-small cell...
Hotspot mutation and fusion transcript detection from the same non-small cell...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.Orthogonal Verification of Oncomine cfDNA Data with Digital PCR Using TaqMan ...
Orthogonal Verification of Oncomine cfDNA Data with Digital PCR Using TaqMan ...Thermo Fisher Scientific
The tumor microenvironment (TME) is the intersection between tumor cells and
surrounding non-transformed cells. It contains immune cells, signaling molecules,
stromal and extracellular matrix. Research has shown the TME is often associated
with tumor growth. However, the function and regulatory mechanism of each
constituent is still poorly understood. The presence of PD-L1 is a promising marker
to predict positive response for T cell checkpoint therapy. Current IHC methods to
measure PD-L1 are subjective and highly variable. A higher-throughput and
standardized method that can systematically measure gene expression of cells
present in the TME has emerged to be a more desirable solution.
We applied the OncomineTM Immune Response Research Assay to measure the
expression of 395 genes in non-small cell lung cancer (NSCLC) research samples
from 40 matched FFPE and fresh frozen sample types. This assay covers genes
involved in checkpoint pathway, T cell regulation, cytokine and interferon signaling
pathways, and markers of different tumor infiltrating lymphocyte (TIL) subsets, as
well as tumor markers. With an input requirement of 10 ng of total RNA, libraries
were generated, templated on the Ion ChefTM and sequenced on the Ion S5TM
System. Sequencing data was analyzed and mapped with Torrent Suite Software
and differential expression analysis was conducted with AffymetrixTM Transcriptome
Analysis Console.Gene expression profile of the tumor microenvironment from 40 NSCLC FFPE and ...
Gene expression profile of the tumor microenvironment from 40 NSCLC FFPE and ...Thermo Fisher Scientific
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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.Low Level Somatic Variant Detection by Sanger Sequencing of FFPE Samples for ...
Low Level Somatic Variant Detection by Sanger Sequencing of FFPE Samples for ...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%.Rare Mutation Analysis Using Digital PCR on QuantStudio™ 3D to Verify Ion Amp...
Rare Mutation Analysis Using Digital PCR on QuantStudio™ 3D to Verify Ion Amp...Thermo Fisher Scientific
As personalized cancer care evolves, the patient’s nucleic acid becomes ever so important to provide valuable information regarding their genetic makeup and disease state. Common sample types for these analyses include biopsies, which can be very limited in material making the downstream measurement of more than one analyte rather difficult. Obtaining another biopsy, using a different section or splitting the sample can be problematic because of tumor heterogeneity. Even adjacent areas of the same tumor tissue can result in different RNA/DNA profiles so the ability to isolate multiple analytes from the same sample offer a number of benefits, which include preserving samples and data consistency eliminating any sample to sample variation. As more tests are developed to simultaneously monitor genetic alterations, there is a strong need to efficiently isolate both DNA and RNA from the same starting sample in a format compatible with high-throughput processing.
High-throughput processing to maximize genomic analysis through simultaneous ...
High-throughput processing to maximize genomic analysis through simultaneous ...Thermo Fisher Scientific
Research has shown that circulating tumor DNA (ctDNA) is
informative of tumor load and tumor evolution in both solid and
hematological cancer. The ability to detect mutations in ctDNA
holds the promise for an accurate and non-invasive approach to
assess minimum residual disease as well as treatment
response in the future. However, as ctDNA often makes up only
a small fraction of cell-free DNA recovered from the plasma,
traditional methods of targeted sequencing research often face
a poor signal-to-noise ratio that cannot be overcome with deep
coverage.
Here we present a novel research method that is capable of
detecting ultra-rare mutations at allelic frequency below 0.5%.
This approach leverages target multiplexing capabilities of the
Ion AmpliSeq™ technology with some important modifications
to the sample preparation procedures. The new protocol
requires as little as 20 ng of input DNA and offers a sample-toanswer
turn-around time in under 24 hours. To support the
analysis of this new approach, we have further developed a
novel Bayesian statistics that models the propagation of
potential artifacts introduced during amplification and sampling
effects during sequencing to differentiate false positives
(variants observed in sequencing data that were not present in
input DNA) from true mutations that were present at very low
levels in the original research sample.
We successfully applied this new method to detect spike-in
mutant DNA in both cell line (Coriell GM24385) and cfDNA
samples. Specifically, we demonstrated the detection of 140
COSMIC genomic aberrations found in 23 frequently mutated
genes. In preliminary study, the method achieved greater 90%
sensitivity and specificity.Successful detection of 40 COSMIC hotspot mutations at allelic frequency belo...
Successful detection of 40 COSMIC hotspot mutations at allelic frequency belo...Thermo Fisher Scientific
We have developed a research method to
prepare Ion AmpliSeq libraries directly from small
amounts of biological samples such as whole
blood, saliva, buccal swabs and even FFPE
sections.
• The direct library prep methods were automated
on the Ion Chef reducing user interaction to
loading reagents and samples on to the deck of
the Ion Chef.
• Sequencing metrics were comparable or
improved for the samples used directly without
purification as compared to purified DNA controls.
Additionally, as shown for buccal swab samples,
there was no detrimental effect on variant calling
results.
• Extremely small samples were successfully
processed with this method, which reduces the
total time from sample to sequence to 24 hours.Preparing libraries directly from archived FFPE sections blood, saliva, and b...
Preparing libraries directly from archived FFPE sections blood, saliva, and b...Thermo Fisher Scientific
The presence of certain chromosomal Header
rearrangements and the subsequent fusion
gene derived from translocations has been
implicated in a number of cancers. Hundreds of
translocations have been described in the
literature recently but the need to efficiently
detect and further characterize these
chromosomal translocations is growing
exponentially. The two main methods to identify
and monitor translocations, fluorescent in situ
hybridization (FISH) and comparative genomic
hybridization (CGH) are challenging, labor
intensive, the information obtained is limited,
and sensitivity is rather low. Common sample
types for these analyses are biopsies or small
tumors, which are very limited in material
making the downstream measurement of more
than one analyte rather difficult; obtaining
another biopsy, using a different section or
splitting the sample can raise issues of tumor
heterogeneity. The ability to study mutation
status as well as measuring fusion transcript
expression from the same sample is powerful
because you’re maximizing the information
obtained from a single precious sample and
eliminating any sample to sample variation.
Here we describe the efficient isolation of two
valuable analytes, RNA and DNA, from the
same starting sample without splitting, followed
by versatile and informative downstream
analysis. This methodology has been applied to
FFPE and degraded samples as well as fresh
tissues, cells and blood. DNA and RNA were
recovered from the same non-small cell lung
adenocarcinoma sample and both mutation
analysis, as well as fusion transcript detection
was performed using the Ion Torrent PGM™
platform on the same Ion 318™ chip. Using
10ng of DNA and 10ng of RNA input, we
applied the Ion AmpliSeq™ Colon and Lung
Cancer panel to analyze over 500 COSMIC
mutations in 22 genes and the Ion AmpliSeq™
RNA Lung Fusion panel to detect 40 different
fusion transcripts.Hotspot mutation and fusion transcript detection from the same non-small cell...
Hotspot mutation and fusion transcript detection from the same non-small cell...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.Orthogonal Verification of Oncomine cfDNA Data with Digital PCR Using TaqMan ...
Orthogonal Verification of Oncomine cfDNA Data with Digital PCR Using TaqMan ...Thermo Fisher Scientific
The tumor microenvironment (TME) is the intersection between tumor cells and
surrounding non-transformed cells. It contains immune cells, signaling molecules,
stromal and extracellular matrix. Research has shown the TME is often associated
with tumor growth. However, the function and regulatory mechanism of each
constituent is still poorly understood. The presence of PD-L1 is a promising marker
to predict positive response for T cell checkpoint therapy. Current IHC methods to
measure PD-L1 are subjective and highly variable. A higher-throughput and
standardized method that can systematically measure gene expression of cells
present in the TME has emerged to be a more desirable solution.
We applied the OncomineTM Immune Response Research Assay to measure the
expression of 395 genes in non-small cell lung cancer (NSCLC) research samples
from 40 matched FFPE and fresh frozen sample types. This assay covers genes
involved in checkpoint pathway, T cell regulation, cytokine and interferon signaling
pathways, and markers of different tumor infiltrating lymphocyte (TIL) subsets, as
well as tumor markers. With an input requirement of 10 ng of total RNA, libraries
were generated, templated on the Ion ChefTM and sequenced on the Ion S5TM
System. Sequencing data was analyzed and mapped with Torrent Suite Software
and differential expression analysis was conducted with AffymetrixTM Transcriptome
Analysis Console.Gene expression profile of the tumor microenvironment from 40 NSCLC FFPE and ...
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CFC
- 1. Cell-Free Circulating DNA & RNA From Plasma/Serum, Urine, Follicular Fluid Isolate from 0.1 mL to 30 mL 0.07 0.13 0.23 100 97 92 0 20 40 60 80 100 120 0.0 0.1 0.1 0.2 0.2 0.3 0.3 0.5mL 1mL 2mL %ofLinearity RelativeAmountof5SrRNA Gene Fetal DNA Screening Microarrays Genotyping NGS Biomarker Discovery Methylation Epigenetics Scalable input volumes Rapid, robust & reliable Excellent linearity Inhibitor-free For all downstream applications cfc-DNA cfc-RNA 0.E+00 2.E-08 4.E-08 6.E-08 8.E-08 1.E-07 1.E-07 1.E-07 Norgen Competitor Q Competitor A Competitor E RelativeAmountofmiR-21in Plasma Norgen Biotek Corp. www.norgenbiotek.com