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.
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.
Ion Torrent™ Next Generation Sequencing-Oncomine™ Lung cfDNA assay detected 0...Thermo Fisher Scientific
Study of genetic Information from cell-free (cf) DNA provide valuable opportunities in cancer research and potentially impact future oncology. As an example, liquid biopsy provide a non-invasive and cost effective solution for future compared to traditional biopsy tests. Here we report the application of research based Ion Torrent™ next-generation sequencing (NGS) Oncomine™ cfDNA assays and associated workflow, which is developed to detect somatic variants at low frequency of 0.1% in cfDNA from plasma.
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.
High-throughput processing to maximize genomic analysis through simultaneous ...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.
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.
Detection of somatic mutations at 0.5% frequency from cfDNA and CTC DNA using...Thermo Fisher Scientific
Availability of effective blood screening for tracking of recurrence and
resistance of tumors may improve outcomes in the future. Research
studies suggest that virtually all tumors carry somatic DNA mutations, and
these may serve as biomarkers that may be tracked from blood. The
two well-characterized sources of tumor DNA in blood are circulating
tumor cells (CTC) and cell-free tumor DNA (ctDNA). The abundance of
CTC and/or ctDNA in blood may be very low at critical stages such as
early recurrence or development of resistance. Hence there is great
interest in being able to detect biomarkers at very low frequency from
blood, and in characterizing the relationship between somatic mutations
present in the tumor and those in CTC or ctDNA.
We present a research use only analysis workflow for peripheral
monitoring that enables detection of low frequency variants in blood. We
developed an analysis algorithm, using statistical modeling of next
generation sequencing reads, and optimizing parameters and filters to
enable sensitive and specific detection of somatic mutations at 0.5% allele
ratio. We demonstrate the analysis on a blood sample split into 3 subsamples
comprising normal blood, CTC enriched, and cell-free DNA
(cfDNA) samples.
We used lysis to isolate white blood cells (germline), centrifugation to
extract plasma DNA (cfDNA), while CTC cells were isolated using
Cynvenio LiquidBiopsy™ platform a fully automated antibody-based
solution. We barcoded 3 sub-samples and run them on a single Ion 318™
sequencing chip using Ion AmpliSeq™ Cancer Hotspot Panel (CHPv2),
that enables very deep (~10,000x coverage) and accurate
sequencing. This panel allows interrogation of ~2800 relevant
biomarkers from COSMIC and FDA actionable databases, and denovo
variant detection at ~20,000 genomic positions. Mutations were
annotated using the Oncomine® database in Ion Reporter™ software. The
research assay requires a small amount of input DNA (~10ng), and has a
fast turn around time from extracted DNA to variants of less than 24 hr.
Successful detection of 40 COSMIC hotspot mutations at allelic frequency belo...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.
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.
Ion Torrent™ Next Generation Sequencing-Oncomine™ Lung cfDNA assay detected 0...Thermo Fisher Scientific
Study of genetic Information from cell-free (cf) DNA provide valuable opportunities in cancer research and potentially impact future oncology. As an example, liquid biopsy provide a non-invasive and cost effective solution for future compared to traditional biopsy tests. Here we report the application of research based Ion Torrent™ next-generation sequencing (NGS) Oncomine™ cfDNA assays and associated workflow, which is developed to detect somatic variants at low frequency of 0.1% in cfDNA from plasma.
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.
High-throughput processing to maximize genomic analysis through simultaneous ...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.
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.
Detection of somatic mutations at 0.5% frequency from cfDNA and CTC DNA using...Thermo Fisher Scientific
Availability of effective blood screening for tracking of recurrence and
resistance of tumors may improve outcomes in the future. Research
studies suggest that virtually all tumors carry somatic DNA mutations, and
these may serve as biomarkers that may be tracked from blood. The
two well-characterized sources of tumor DNA in blood are circulating
tumor cells (CTC) and cell-free tumor DNA (ctDNA). The abundance of
CTC and/or ctDNA in blood may be very low at critical stages such as
early recurrence or development of resistance. Hence there is great
interest in being able to detect biomarkers at very low frequency from
blood, and in characterizing the relationship between somatic mutations
present in the tumor and those in CTC or ctDNA.
We present a research use only analysis workflow for peripheral
monitoring that enables detection of low frequency variants in blood. We
developed an analysis algorithm, using statistical modeling of next
generation sequencing reads, and optimizing parameters and filters to
enable sensitive and specific detection of somatic mutations at 0.5% allele
ratio. We demonstrate the analysis on a blood sample split into 3 subsamples
comprising normal blood, CTC enriched, and cell-free DNA
(cfDNA) samples.
We used lysis to isolate white blood cells (germline), centrifugation to
extract plasma DNA (cfDNA), while CTC cells were isolated using
Cynvenio LiquidBiopsy™ platform a fully automated antibody-based
solution. We barcoded 3 sub-samples and run them on a single Ion 318™
sequencing chip using Ion AmpliSeq™ Cancer Hotspot Panel (CHPv2),
that enables very deep (~10,000x coverage) and accurate
sequencing. This panel allows interrogation of ~2800 relevant
biomarkers from COSMIC and FDA actionable databases, and denovo
variant detection at ~20,000 genomic positions. Mutations were
annotated using the Oncomine® database in Ion Reporter™ software. The
research assay requires a small amount of input DNA (~10ng), and has a
fast turn around time from extracted DNA to variants of less than 24 hr.
Successful detection of 40 COSMIC hotspot mutations at allelic frequency belo...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.
Preparing libraries directly from archived FFPE sections blood, saliva, and b...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.
Sequencing the circulating and infiltrating T-cell repertoire on the Ion S5TMThermo Fisher Scientific
T-Cell receptor (TCR) repertoire sequencing by next-generation
sequencing (NGS) is a valuable tool for building a deeper
understanding of the adaptive immune system. As immunotherapy,
particularly T-cell therapies, show increasing potential in treating
cancer, the ability to gain a detailed, unbiased view of the TCR
repertoire becomes imperative for biomarker discovery, immune
response to treatment, and study of tumor microenvironments. A key
question the field seeks to understand is the relationship between
circulating T-cells and infiltrating T-cells at the tumor site. Here, we
present a novel AmpliSeq approach for TCR repertoire sequencing
using the Ion Torrent S5 sequencer which leverages simplified
workflows and offers up to 600 bp reads which allow for a more
complete characterization of the entire V(D)J region of TCRβ. With a
unique long read length capability, this method can leverage mRNA as
input, which minimizes requirement as starting materials (10-500ng for
typical use cases) and focusing sequencing to productive TCRβ
arrangements.
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.
600 base reads on the Ion S5™ Next-Generation Sequencing System enables accur...Thermo Fisher Scientific
Longer read lengths simplify genome assembly,
haplotyping, metagenomics, and the design of library
primers for targeted resequencing. Several new
technologies were developed to enable the
sequencing of templates with inserts over 600 bases:
a fast isothermal templating technology, an ISP™
that is optimized for maximum template density, a
new long-read sequencing polymerase, and
instrument scripts that consume less reagents. We
demonstrate the combination of these technologies
to sequence 600 base long DNAs on an Ion 530
Chip™ with an average AQ20 mean read length over
500 bp. The protocol was used to type human
leukocyte antigen (HLA) alleles, a haplotyping
application that is greatly simplified by long read
length sequence data. 96 HLA samples were typed
with 99.7% concordance to truth on one Ion 530
chip.
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.
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.
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.
Gene expression profile of the tumor microenvironment from 40 NSCLC FFPE and ...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.
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.
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.
The enzyme Telomerase maintains telomeres at the ends of
chromosomes. The Telomerase Reverse Transcriptase (TERT)
gene codes for the enzyme’s catalytic domain and is not
expressed in normal somatic cells. As a consequence, normal
cells acquire senescence by shortening of their telomeres
during cell division and eventually undergo apoptosis. In
contrast to normal somatic cells, expression of TERT is
reinstated in cancer cells causing escape from senescence and
apoptosis by maintaining the telomeres. It has recently been
shown that mutations in the TERT promoter region play a key
role in regulating and reinstating TERT expression. Up to 90%
of cancers carry a mutation in the TERT promoter region.
Mutations like C228T and C250T create new binding sites for
the E26 transformation-specific (ETS) transcription factor that
regulates TERT expression (1,2). Experimental evidence
showed that the ETS factor GA-binding protein, alpha subunit
(GABPA) binds to the de novo ETS motif and activates TERT
transcription in cancer cells.
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%.
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.
Comparing Mutation Detection Sensitivity from Matched FFPE Tissue and Liquid ...Thermo Fisher Scientific
Cancer researchers are avidly working to enable circulating cell free DNA (cfDNA) profiling as a new more sensitive tool to detect and screen for the presence of solid tumors before detection through clinical methods. Despite the high level of interest in cfDNA, researchers still have reservations until enough data has demonstrated complementarity between methodologies. In this study, we examined the data quality and concordance of mutations called for a small number of matched formalin fixed paraffin embedded (FFPE) tissue and plasma samples.
An Efficient NGS Workflow for Liquid Biopsy Research Using a Comprehensive As...Thermo Fisher Scientific
Recent studies in non-invasive biomarker research have demonstrated the potential of using cell-free nucleic acids isolated from blood plasma to serve as surrogates for solid tumors. Somatic mutations representing the tumors could be successfully detected from cell-free DNA (cfDNA) and cell-free RNA (cfRNA), providing new tumor assessment methods in addition to tissue biopsy. However, the low amount of circulating tumor fragments in the blood presents significant challenges for accurate variant detection with NGS assays. Moreover, utilization of both cfDNA and cfRNA requires methods capable of interrogating both types of analytes to maximize the utility of each blood sample.
Localized gene expression changes by AmpliSeq transcriptome sequencing from A...Thermo Fisher Scientific
We have developed a simplified workflow that enables unbiased
transcriptome analysis of specifically selected cells from fresh frozen or
FFPE archived brain samples. Archival frozen sections and FFPE
sections of temporal lobes from Alzheimer-affected and normal brains
were obtained from a commercial source. Regions of brain tissue were
collected using an ArcturusXT system and RNA eluted from these was
sequenced for gene expression of the transcriptome (~20,000 genes).
LCM allowed us to detect differences in expression patterns in
morphologically distinct regions of the brain as well as detect patterns of
expression related to the Alzheimer pathology iin the immediate vicinity of
pathogenic plaques and tangles.
Finally, we show that using LCM to enrich cells around the plaques and
tangles enabled the detection of gene expression changes that were
undetectable in whole tissue scrapes. This workflow will enable
researchers to gain novel insights into questions requiring analysis of gene
expression patterns of extremely discrete cell populations in an otherwise
heterogeneous tissue source.
Simple, rapid preparation of genomic libraries from single human cells and ba...Thermo Fisher Scientific
MuSeek is a MuA transposon-based library preparation
method that offers a streamlined workflow and the sensitivity
necessary for low input experiments. We are exploring
improvements that reduce required processing steps and
enhance performance.
We present data using cell lysates as direct input into the
library preparation reaction:
• A single bacterial colony for whole genome sequencing
(WGS)
• Single human cells for low-pass aneuploidy detection
These improvements to MuSeek offer exciting new
possibilities for simple, rapid, and low input library preparation
up front of the Ion Torrent™ Sequencing Systems.
Development of a Breast and Lung Cancer Research Panel To Target Therapeutica...Thermo Fisher Scientific
With recent advances in next-generation sequencing (NGS) technologies, it is now possible to detect somatic mutations with allele frequencies in blood samples as low as 0.1% from circulating tumor DNA. A natural extension to this achievement is adding the ability to simultaneously detect copy number variants and gene fusions. A panel such as this addresses a full repertoire of variant classes found to be linked with certain tumors and would enable researchers additional to profile cancer samples more dynamically thus enriching current diagnostic tool sets. Here, we present progress on such an approach and apply current NGS technology to achieve our goals.
Cystic Fibrosis is an autosomal recessive genetic disease that is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which has important roles in ion exchange.
Forecasting clinical behavior and therapeutic response of human cancer currently utilizes a limited number of tumor markers in combination with characteristics of the patient and their disease. Although few tumor markers and molecular targets exist for evaluation, the wealth of information derived from recent sequencing advancements provides greater opportunities to develop more precise tests for diagnostics, prognostics, therapy selection and monitoring in the future. The objectives of this study are to study miRNA and mRNA expression profiles of laser capture microdissection (LCM)-procured tumor cells and intact serial sections of breast tissue samples using next generation sequencing (NGS) methods. Our hypothesis is that miRNA signatures discerned from specific tumor cell populations more precisely correlate with behavior than that provided by conventional biomarkers from intact tissue samples. Additionally, we hypothesize the data generated in this study will present mRNA signatures informative for breast tumor research and support our miRNA findings through suggesting relevant miRNA:mRNA target associations.
De-identified frozen research samples of primary invasive ductal tumors of known grade and biomarker status containing 35-70% tumor were selected from an IRB-approved Biorepository. Comparison of expressed miRNAs from intact tissue sections with those of cognate tumor cells procured by LCM revealed, in general, that smaller defined miRNA gene sets were expressed in LCM isolated populations of tumor cells. In addition to miRNA sequencing, targeted RNA sequencing with the Ion AmpliSeq™ Transcriptome Human Gene Expression Kit was used to capture mRNA expression information. Data presented here demonstrates high mapping rates for targeted mRNA (>91% of reads) and miRNA (> 88% of reads) libraries. We also demonstrate high technical reproducibility between multiple libraries from the same tumor sample for both mRNA (R>0.99) and miRNA (R>0.97) libraries. We also report suggested miRNA:mRNA target associations identified in our set of breast tumor research samples. These data provide insights into breast cancer biology that may lead to new molecular diagnostics and targets for drug design in the future as well as an improved understanding of the molecular basis of clinical behavior and potential therapeutic response.
Rise above the risk of the helium supply shortage with innovative solutions that optimize the usage of the non-renewable gas. The new Thermo Scientific™ Instant Connect Helium Saver Module enables you to continue your analyses without helium supply concerns or extensive supply costs.
Save precious non-renewable noble helium when your instrument is idle, as well as during the analytical run. Keep your methods intact, validation-effective, and regulation-compliant. Find out how to extend the lifetime of one helium cylinder to over 14 years under certain conditions and experience significant savings in both helium supply needs and financial costs throughout the lifetime of your Gas Chromatography-Mass Spectrometry instrument.
· Maintain your methods
· Prolong your helium supply
· Save your budget
Preparing libraries directly from archived FFPE sections blood, saliva, and b...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.
Sequencing the circulating and infiltrating T-cell repertoire on the Ion S5TMThermo Fisher Scientific
T-Cell receptor (TCR) repertoire sequencing by next-generation
sequencing (NGS) is a valuable tool for building a deeper
understanding of the adaptive immune system. As immunotherapy,
particularly T-cell therapies, show increasing potential in treating
cancer, the ability to gain a detailed, unbiased view of the TCR
repertoire becomes imperative for biomarker discovery, immune
response to treatment, and study of tumor microenvironments. A key
question the field seeks to understand is the relationship between
circulating T-cells and infiltrating T-cells at the tumor site. Here, we
present a novel AmpliSeq approach for TCR repertoire sequencing
using the Ion Torrent S5 sequencer which leverages simplified
workflows and offers up to 600 bp reads which allow for a more
complete characterization of the entire V(D)J region of TCRβ. With a
unique long read length capability, this method can leverage mRNA as
input, which minimizes requirement as starting materials (10-500ng for
typical use cases) and focusing sequencing to productive TCRβ
arrangements.
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.
600 base reads on the Ion S5™ Next-Generation Sequencing System enables accur...Thermo Fisher Scientific
Longer read lengths simplify genome assembly,
haplotyping, metagenomics, and the design of library
primers for targeted resequencing. Several new
technologies were developed to enable the
sequencing of templates with inserts over 600 bases:
a fast isothermal templating technology, an ISP™
that is optimized for maximum template density, a
new long-read sequencing polymerase, and
instrument scripts that consume less reagents. We
demonstrate the combination of these technologies
to sequence 600 base long DNAs on an Ion 530
Chip™ with an average AQ20 mean read length over
500 bp. The protocol was used to type human
leukocyte antigen (HLA) alleles, a haplotyping
application that is greatly simplified by long read
length sequence data. 96 HLA samples were typed
with 99.7% concordance to truth on one Ion 530
chip.
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.
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.
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.
Gene expression profile of the tumor microenvironment from 40 NSCLC FFPE and ...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.
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.
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.
The enzyme Telomerase maintains telomeres at the ends of
chromosomes. The Telomerase Reverse Transcriptase (TERT)
gene codes for the enzyme’s catalytic domain and is not
expressed in normal somatic cells. As a consequence, normal
cells acquire senescence by shortening of their telomeres
during cell division and eventually undergo apoptosis. In
contrast to normal somatic cells, expression of TERT is
reinstated in cancer cells causing escape from senescence and
apoptosis by maintaining the telomeres. It has recently been
shown that mutations in the TERT promoter region play a key
role in regulating and reinstating TERT expression. Up to 90%
of cancers carry a mutation in the TERT promoter region.
Mutations like C228T and C250T create new binding sites for
the E26 transformation-specific (ETS) transcription factor that
regulates TERT expression (1,2). Experimental evidence
showed that the ETS factor GA-binding protein, alpha subunit
(GABPA) binds to the de novo ETS motif and activates TERT
transcription in cancer cells.
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%.
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.
Comparing Mutation Detection Sensitivity from Matched FFPE Tissue and Liquid ...Thermo Fisher Scientific
Cancer researchers are avidly working to enable circulating cell free DNA (cfDNA) profiling as a new more sensitive tool to detect and screen for the presence of solid tumors before detection through clinical methods. Despite the high level of interest in cfDNA, researchers still have reservations until enough data has demonstrated complementarity between methodologies. In this study, we examined the data quality and concordance of mutations called for a small number of matched formalin fixed paraffin embedded (FFPE) tissue and plasma samples.
An Efficient NGS Workflow for Liquid Biopsy Research Using a Comprehensive As...Thermo Fisher Scientific
Recent studies in non-invasive biomarker research have demonstrated the potential of using cell-free nucleic acids isolated from blood plasma to serve as surrogates for solid tumors. Somatic mutations representing the tumors could be successfully detected from cell-free DNA (cfDNA) and cell-free RNA (cfRNA), providing new tumor assessment methods in addition to tissue biopsy. However, the low amount of circulating tumor fragments in the blood presents significant challenges for accurate variant detection with NGS assays. Moreover, utilization of both cfDNA and cfRNA requires methods capable of interrogating both types of analytes to maximize the utility of each blood sample.
Localized gene expression changes by AmpliSeq transcriptome sequencing from A...Thermo Fisher Scientific
We have developed a simplified workflow that enables unbiased
transcriptome analysis of specifically selected cells from fresh frozen or
FFPE archived brain samples. Archival frozen sections and FFPE
sections of temporal lobes from Alzheimer-affected and normal brains
were obtained from a commercial source. Regions of brain tissue were
collected using an ArcturusXT system and RNA eluted from these was
sequenced for gene expression of the transcriptome (~20,000 genes).
LCM allowed us to detect differences in expression patterns in
morphologically distinct regions of the brain as well as detect patterns of
expression related to the Alzheimer pathology iin the immediate vicinity of
pathogenic plaques and tangles.
Finally, we show that using LCM to enrich cells around the plaques and
tangles enabled the detection of gene expression changes that were
undetectable in whole tissue scrapes. This workflow will enable
researchers to gain novel insights into questions requiring analysis of gene
expression patterns of extremely discrete cell populations in an otherwise
heterogeneous tissue source.
Simple, rapid preparation of genomic libraries from single human cells and ba...Thermo Fisher Scientific
MuSeek is a MuA transposon-based library preparation
method that offers a streamlined workflow and the sensitivity
necessary for low input experiments. We are exploring
improvements that reduce required processing steps and
enhance performance.
We present data using cell lysates as direct input into the
library preparation reaction:
• A single bacterial colony for whole genome sequencing
(WGS)
• Single human cells for low-pass aneuploidy detection
These improvements to MuSeek offer exciting new
possibilities for simple, rapid, and low input library preparation
up front of the Ion Torrent™ Sequencing Systems.
Development of a Breast and Lung Cancer Research Panel To Target Therapeutica...Thermo Fisher Scientific
With recent advances in next-generation sequencing (NGS) technologies, it is now possible to detect somatic mutations with allele frequencies in blood samples as low as 0.1% from circulating tumor DNA. A natural extension to this achievement is adding the ability to simultaneously detect copy number variants and gene fusions. A panel such as this addresses a full repertoire of variant classes found to be linked with certain tumors and would enable researchers additional to profile cancer samples more dynamically thus enriching current diagnostic tool sets. Here, we present progress on such an approach and apply current NGS technology to achieve our goals.
Cystic Fibrosis is an autosomal recessive genetic disease that is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which has important roles in ion exchange.
Forecasting clinical behavior and therapeutic response of human cancer currently utilizes a limited number of tumor markers in combination with characteristics of the patient and their disease. Although few tumor markers and molecular targets exist for evaluation, the wealth of information derived from recent sequencing advancements provides greater opportunities to develop more precise tests for diagnostics, prognostics, therapy selection and monitoring in the future. The objectives of this study are to study miRNA and mRNA expression profiles of laser capture microdissection (LCM)-procured tumor cells and intact serial sections of breast tissue samples using next generation sequencing (NGS) methods. Our hypothesis is that miRNA signatures discerned from specific tumor cell populations more precisely correlate with behavior than that provided by conventional biomarkers from intact tissue samples. Additionally, we hypothesize the data generated in this study will present mRNA signatures informative for breast tumor research and support our miRNA findings through suggesting relevant miRNA:mRNA target associations.
De-identified frozen research samples of primary invasive ductal tumors of known grade and biomarker status containing 35-70% tumor were selected from an IRB-approved Biorepository. Comparison of expressed miRNAs from intact tissue sections with those of cognate tumor cells procured by LCM revealed, in general, that smaller defined miRNA gene sets were expressed in LCM isolated populations of tumor cells. In addition to miRNA sequencing, targeted RNA sequencing with the Ion AmpliSeq™ Transcriptome Human Gene Expression Kit was used to capture mRNA expression information. Data presented here demonstrates high mapping rates for targeted mRNA (>91% of reads) and miRNA (> 88% of reads) libraries. We also demonstrate high technical reproducibility between multiple libraries from the same tumor sample for both mRNA (R>0.99) and miRNA (R>0.97) libraries. We also report suggested miRNA:mRNA target associations identified in our set of breast tumor research samples. These data provide insights into breast cancer biology that may lead to new molecular diagnostics and targets for drug design in the future as well as an improved understanding of the molecular basis of clinical behavior and potential therapeutic response.
Rise above the risk of the helium supply shortage with innovative solutions that optimize the usage of the non-renewable gas. The new Thermo Scientific™ Instant Connect Helium Saver Module enables you to continue your analyses without helium supply concerns or extensive supply costs.
Save precious non-renewable noble helium when your instrument is idle, as well as during the analytical run. Keep your methods intact, validation-effective, and regulation-compliant. Find out how to extend the lifetime of one helium cylinder to over 14 years under certain conditions and experience significant savings in both helium supply needs and financial costs throughout the lifetime of your Gas Chromatography-Mass Spectrometry instrument.
· Maintain your methods
· Prolong your helium supply
· Save your budget
Recombinant Expression and Purification of Aedes aegypti Midgut Serine Protea...Kamille Parungao
The Aedes aegypti mosquito is a major vector of blood-borne pathogens, such as the Dengue, Chikungunya, yellow fever, and Zika viruses. This poster discusses the recombinant expression and purification of a late-phase trypsin- like protease, Aedes aegypti serine protease VII (AaSPVII).
Data-independent acquisition (DIA) is becoming increasingly important for quantitative proteomics, especially for large-scale targeted protein quantification. DIA provides better reproducibility, acquires fragment ions of all precursors, and breaks through the limit of quantification throughput. DIA with the Thermo Scientific™ Orbitrap™ LC-MS enables ultimate flexibility. The Thermo Scientific™ Orbitrap™ LC-MS pushes the boundaries of large scale quantitation, with highest mass accuracy, Orbitrap high resolution, short cycle times, and narrow isolation windows. Learn more at www.thermoscientific.com/DIAwebinar
It was my OB presentation on Thermo Fisher scientific ltd. We have covered journey of Thermo Fisher, Organisation structure, Organisation configuration, Organisation Design, Integrated Model, Relationship with Organizations, Culture etc topics.
SmashFly Transform: How Storytelling Transformed Thermo Fisher's Employer BrandSmashFly Technologies
This is the way to give an employer brand presentation: by leading with employees and their stories. Charlotte Marshall, Employer Brand Leader at Thermo Fisher Scientific, humanized and personalized the company's employer brand by telling the authentic stories of the people who make the company successful every day.
Presentation given at the Stockholm R useR Group (SRUG) meetup on Dec 6, 2016. Contains a general overview of deep learning, material on using Tensorflow in R etc.
AWS re:Invent 2016: How Thermo Fisher Is Reducing Mass Spectrometry Experimen...Amazon Web Services
Mass spectrometry is the gold standard for determining chemical compositions, with spectrometers often measuring the mass of a compound down to a single electron. This level of granularity produces an enormous amount of hierarchical data that doesn't fit well into rows and columns. In this talk, learn how Thermo Fisher is using MongoDB Atlas on AWS to allow their users to get near real-time insights from mass spectrometry experiments—a process that used to take days. We also share how the underlying database service used by Thermo Fisher was built on AWS.
While Europe might be a single market, it’s definitely not a single tech scene. That fact makes it difficult to feel the pulse of the European tech. But if you take a close look, you’ll quickly notice that there is interesting stuff happening in Europe
With this report we want to provide a comprehensive review of investment in startups and high-growth technology companies across 31 countries in Europe. Our aim is to provide data-driven guidance, insights and inspiration to stakeholders in the European scaleup ecosystem.
Recommendations on ISR in multi‐analyte assays, QA/bioanalytical consultants ...Wei Garofolo
The 5th Global CRO Council for Bioanalysis (GCC) meeting, held in Barcelona, Spain, in November 2011, provided a unique opportunity for CRO leaders to openly share opinions, perspectives and to agree on bioanalytical recommendations on incurred sample reproducibility in multi-analyte assays, regulation of quality assurance/bioanalytical consultants and regulatory requirements for GCP.
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.
Development of cell culture samples for drug screening with bone marrow stem ...Apollo Hospitals
Side population (SP) refers to a group of cells, which is capable to efflux Hoechst 33342, a DNA-binding dye. SP cells exist both in normal and tumor tissues. SP abundance is used as an indicator for disease prognosis and drug screening in some studies. Our study concentrates on the factors influencing SP analysis.
Monitoring histological changes in oral mucosa using AgNORs as biomarkers for...iosrphr_editor
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
TEMPUS xT, Biópsia sólida vs TEMPUS xF, Biópsia LíquidaComunicaoIberlab
Para amostras correspondentes, a concordância do teste Tempus xF em comparação com o Tempus xT para alvos clinicamente acionáveis foi de 74,31%.
65% dos pacientes descobriram pelo menos uma variante patogénica extra quando a biópsia líquida e o tumor sólido foram solicitados simultaneamente, em comparação apenas com o tumor sólido.
Assessment of TP53 Mutation Status in Breast Tumor Tissue using the "Ion Ampl...Thermo Fisher Scientific
TP53 mutations are found in 30% of breast tumors and are associated with poor prognosis in distinct subtypes of breast cancer. Direct sequencing is commonly used to obtain TP53 mutation status in tumor tissue, but has limitations in detection level and is time-consuming. Methods targeting hotspots is insufficient for TP53 analysis since the mutations are widely spread along the gene1. Here we describe the development of the Ion TorrentTM next-generation semiconductor sequencing and Ion AmpliSeqTM technology (Life TechnologiesTM).
Parameter Optimization of Shot Peening Process of PMG AL2024 Alloy CoverIOSRJMCE
Shot peening leads to local plastic deformations in the near-surface regions, which result in the development of compressive residual stress and the improvement of surface hardness in the aerospace structural components. These properties can be enhanced by careful selection of the peening parameters. PMG Cover of AL2024 Aluminum Alloy is widely used in the generator manufacturing cover due to its high specific static strength. In this study a Taguchi Grey Relational Analysis is presented to optimize the surface properties of residual stress, micro hardness. The effects of four peening parameters (Shot Diameter, Shot Velocity, Impact Angle, Nozzle Distance) on micro hardness and residual stress are investigated Design of Experiment work is carried out by MINITAB 14 software tools of Taguchi Grey relational method, for getting excellent shot peening process parameter combination by MAT LAB R2009 software tools of advanced Optimization method as Genetic Algorithm, Simulated Annealing. Compare of the above reading for the investigation.
Clinical Trial Simulation to Evaluate the Pharmacokinetics of an Abuse-Deterr...Loan Pham
A CTS was performed to estimate the sample size and optimal plasma sampling times that sufficiently characterize the PK parameters (CL, Vd) from a single dose of an abuse-deterrent (AD) opioid in pediatric subjects.
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
Human cytomegalovirus (CMV) is a common immune-evasive herpes family virus leading to lifelong asymptomatic infection in 50 to 80% of humans. Current research evaluating the use of
TCR sequencing to predict response to immunotherapy has focused on measurements of T cell clonal expansion and TCR convergence (2,3,4) as potential predictive biomarkers for
response. Given that CMV infection has been reported to elicit large clonal proliferations of CMV reactive T cells (1), and is a source of chronic antigen stimulation, we hypothesized that CMV
infection might alter T cell repertoire features in a manner relevant to the potential biomarker use of TCR sequencing. Here we sought to identify features of CMV infection using TCRB profiling of
peripheral blood (PBL) total RNA. We identify reduced T cell evenness and elevated TCR convergence as features of chronic CMV infection.
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.
What can we learn from oncologists? A survey of molecular testing patternsThermo Fisher Scientific
Oncologists are increasingly incorporating NGS testing to guide targeted and immuno-oncology therapies1. Most clinical NGS testing is confined to large academic institutions and reference labs, despite the fact that most cancer patients are treated in the community settings. We therefore sought to examine molecular testing selection patterns directly from oncologists in order to better identify perceived gaps in testing and treatment paradigms
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.
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.
Novel Spatial Multiplex Screening of Uropathogens Associated with Urinary Tra...Thermo Fisher Scientific
Accurate identification of uropathogens in a timely manner is important to correctly understand urinary tract infections(UTI’s), which affects nearly 150 million people each year. The
current standard approach for detecting the UTI pathogens is culture based. This method is time consuming, has low throughput, and can lack sensitivity and/or specificity. In addition, not all uropathogens grow equally well under standard culture conditions which can result in a failure to detect the species. To address these gaps, we have developed a unique workflow from sample preparation to target identification using the nanofluidic OpenArray™ platform for spatial multiplexing of target specific assays. In this study, we tested pre-determined blinded research samples and confirmed the subset of results with orthogonal Sanger sequences.
Liquid biopsy quality control – the importance of plasma quality, sample prep...Thermo Fisher Scientific
Liquid biopsy is emerging as a non-invasive companion to traditional solid tumor biopsies. As next generation sequencing (NGS) of circulating cell-free nucleic acids (cfNA = cfDNA and cfRNA) becomes common, it’s important to understand the impact of sample preparation on quality, specificity, and sensitivity of liquid biopsy tests. Plasma samples are often limited, and may have undesirable characteristics such as lipemia or hemolysis that contribute unwanted genomic DNA (gDNA) to the sample. Low cfDNA concentration can also limit the amount available for NGS library prep. In this study, we explore the effects of suboptimal plasma and low library input on liquid biopsy NGS, and discuss various techniques for in-process quality control of cfNA samples isolated from plasma
Streamlined next generation sequencing assay development using a highly multi...Thermo Fisher Scientific
Next generation sequencing (NGS) assay development for solid tumor sequencing requires characterization of variant calling directly from formalin-fixed paraffin embedded (FFPE) tissue samples. However, cell line based FFPE and human FFPE samples only contain 2 to 20 variants, which require laboratories to invest significant resources in sample sourcing and preparation when developing assays to detect 100+ variants
Targeted T-cell receptor beta immune repertoire sequencing in several FFPE ti...Thermo Fisher Scientific
T-cell receptor beta (TCRβ) immune repertoire analysis by next-generation sequencing is a valuable tool for studies of the tumor microenvironment and potential immune responses to cancer immunotherapy. Here we describe a TCRβ sequencing assay that leverages the low sample input requirements of AmpliSeq library preparation technology to extend the capability of targeted immune repertoire sequencing to include FFPE samples which can often be degraded and in short supply
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.
As one of the leading causes of death globally, respiratory
infections could be caused by single or multiple types of viral,
bacterial or fungal pathogens that present in the upper and
lower respiratory tract. Panel-based testing using molecular
methods to identify multiple pathogens simultaneously can
contribute to better understanding of respiratory infections.
A high-throughput approach for multi-omic testing for prostate cancer researchThermo Fisher Scientific
The proliferation of genetic testing technologies and genome-scale studies has increased our understanding of the genetic basis of complex diseases. However, this information alone tells an incomplete story of the underlying biology. Integrative approaches that combine data from multiple sources, such as the genome, transcriptome and/or proteome, can provide a more comprehensive and multi-dimensional model of complex diseases. Similarly, the integration of multiple data types in disease screening can improve our understanding of disease in populations. In a series of groundbreaking multi-omic, population-based studies of prostate cancer, researchers at the Karolinska Institutet in Stockholm, Sweden identified sets of genetic and protein biomarkers that when evaluated together with other clinical research data performed significantly better in predicting cancer risk (1,2) than the most-widely used single protein biomarker, the prostate-specific antigen (PSA).
Discover the innovations and more that led to amazing discoveries through the use of thermal cyclers. What were scientists able to accomplish? What things are important to them when selecting a thermal cycler? What do you need to advance your science?
Learn more about thermal cyclers: http://bit.ly/2Q2oPhF
See all thermal cycler offerings: http://bit.ly/2Paf1wH
A rapid library preparation method with custom assay designs for detection of...Thermo Fisher Scientific
Herein, we describe a new research method for library
preparation using the Ion AmpliSeq™ HD Library Kit with
custom assay designs from Ion AmpliSeq HD Panels for
detection of low level variants from liquid biopsy samples. This
method includes incorporation of molecular tags that enable
0.1% Limit of Detection (LOD) in cell free DNA (cfDNA) and
dual barcodes for sample identification. This method is also
applicable to formalin-fixed paraffin embedded (FFPE)
samples. The libraries can be prepared in as little as 3 hours
and are compatible for analysis with the Ion GeneStudio™ S5
system
Generation of Clonal CRISPR/Cas9-edited Human iPSC Derived Cellular Models an...Thermo Fisher Scientific
Reprogramming permits the derivation of hiPSCs from diseased patients, and allows us to model diseases in vitro. Furthermore, with the advent of CRISPR mediated genome editing, we can now mimic disease mutations in control hiPSC lines to study the biological effect of just those mutations. hiPSCs can then be differentiated into specified cell types such as neurons which can be used to develop assays for drug safety screening or can be used to model disease phenotypes in a dish to discover new drugs.
TaqMan®Advanced miRNA cDNA synthesis kit to simultaneously study expression o...Thermo Fisher Scientific
MicroRNAs (miRNA) are a class of small non-coding RNAs (approximately 21 nt long) that bind complementary sequences in target mRNAs to specifically regulate gene expression. Aberrant regulation of miRNAs and their targets has been associated with several diseases including cancer. The relationship between miRNA and mRNA has been found to be important in cancer development and progression. Simultaneous expression studies of miRNA and mRNA and detection of mutations in mRNA transcripts can be valuable in understanding molecular mechanisms that
have an underlying role in various diseases. We demonstrate the technical verification of a novel method to reverse-transcribe and pre-amplify miRNA and mRNA from sample-limiting serum research samples using the TaqMan® Advanced miRNA cDNA Synthesis Kit. Based on results from previous studies, a signature of 49 mRNA and 37 miRNA targets has been identified that may help distinguish between benign and malignant pancreatic tissues. In this study, these targets and an additional set of transcript mutations were analyzed in serum from normal and test samples. TaqMan assays for miRNA and mRNA targets and custom TaqMan Mutation Detection Assays (TMDAs) were placed on TaqMan Array Cards to facilitate investigation of several samples in a single experiment. Results demonstrate that transcript mutations can be detected and miRNA and mRNA targets can be reliably quantified from a single reverse transcription reaction. For research use only. Not for use in diagnostic purposes.
Identifying novel and druggable targets in a triple negative breast cancer ce...Thermo Fisher Scientific
In this study, we developed a CRISPR/Cas9-based high throughput loss-of-function screen for identifying target genes responsible for the tumor proliferation and growth in TNBC. Our initial focus was to identify essential kinases in MDA-MB-231 cell line using the Invitrogen™ LentiArray™ Human Kinase CRISPR Library, which targets 840 kinases with up to 4 different gRNAs per protein kinase for complete gene knockout. This functional screen identified over 90 protein kinases that are essential for cell viability and cell proliferation. Ten of these hits (CDK1, CDK2, CDK8, CDK10, CDK11A, CDK19, CDK19, CDC7, EPHA2 and WEE1) are well-known targets validated in the literature. Currently, we are in the process validating the novel hits through target gene sequencing, western blotting and target specific small molecule kinase inhibitors.
Evidence for antigen-driven TCRβ chain convergence in the melanoma-infiltrati...Thermo Fisher Scientific
T cell convergence refers to the phenomenon whereby antigen-driven selection enriches for T cell receptors (TCRs) having a shared antigen specificity but different amino acid or
nucleotide sequence. T cell recruitment and expansion within the tumor microenvironment (TME) may be directed by responses to tumor neoantigen, suggesting that elevated T
cell convergence could be a general feature of the tumor infiltrating T cell repertoire. Here we use the Ion AmpliSeq™ Immune Repertoire Assay Plus – TCRβ to evaluate evidence
for T cell convergence within melanoma tumor biopsy research samples from a set of 63 subjects plus peripheral blood leukocytes (PBL) from four healthy subjects. We find that the melanoma TME is highly enriched for convergent TCRs compared to healthy donor peripheral blood. We discuss the potential use of TCR convergence as a liquid biopsy compatible predictive biomarker for immunotherapy response.
Analytical performance of a novel next generation sequencing assay for Myeloi...Thermo Fisher Scientific
To support clinical and translational research into precision oncology strategies for myeloid cancers, a next-generation sequencing (NGS) assay was developed to detect common and relevant somatic alterations. To define gene targets that were recurrently altered in myeloid cancers and relevant for clinical and translational research, an extensive survey of investigators at hematology oncology research labs was performed.
The increased availability of biomedical data, particularly in the public domain, offers the opportunity to better understand human health and to develop effective therapeutics for a wide range of unmet medical needs. However, data scientists remain stymied by the fact that data remain hard to find and to productively reuse because data and their metadata i) are wholly inaccessible, ii) are in non-standard or incompatible representations, iii) do not conform to community standards, and iv) have unclear or highly restricted terms and conditions that preclude legitimate reuse. These limitations require a rethink on data can be made machine and AI-ready - the key motivation behind the FAIR Guiding Principles. Concurrently, while recent efforts have explored the use of deep learning to fuse disparate data into predictive models for a wide range of biomedical applications, these models often fail even when the correct answer is already known, and fail to explain individual predictions in terms that data scientists can appreciate. These limitations suggest that new methods to produce practical artificial intelligence are still needed.
In this talk, I will discuss our work in (1) building an integrative knowledge infrastructure to prepare FAIR and "AI-ready" data and services along with (2) neurosymbolic AI methods to improve the quality of predictions and to generate plausible explanations. Attention is given to standards, platforms, and methods to wrangle knowledge into simple, but effective semantic and latent representations, and to make these available into standards-compliant and discoverable interfaces that can be used in model building, validation, and explanation. Our work, and those of others in the field, creates a baseline for building trustworthy and easy to deploy AI models in biomedicine.
Bio
Dr. Michel Dumontier is the Distinguished Professor of Data Science at Maastricht University, founder and executive director of the Institute of Data Science, and co-founder of the FAIR (Findable, Accessible, Interoperable and Reusable) data principles. His research explores socio-technological approaches for responsible discovery science, which includes collaborative multi-modal knowledge graphs, privacy-preserving distributed data mining, and AI methods for drug discovery and personalized medicine. His work is supported through the Dutch National Research Agenda, the Netherlands Organisation for Scientific Research, Horizon Europe, the European Open Science Cloud, the US National Institutes of Health, and a Marie-Curie Innovative Training Network. He is the editor-in-chief for the journal Data Science and is internationally recognized for his contributions in bioinformatics, biomedical informatics, and semantic technologies including ontologies and linked data.
Seminar of U.V. Spectroscopy by SAMIR PANDASAMIR PANDA
Spectroscopy is a branch of science dealing the study of interaction of electromagnetic radiation with matter.
Ultraviolet-visible spectroscopy refers to absorption spectroscopy or reflect spectroscopy in the UV-VIS spectral region.
Ultraviolet-visible spectroscopy is an analytical method that can measure the amount of light received by the analyte.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
Monitor common gases, weather parameters, particulates.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
Nutraceutical market, scope and growth: Herbal drug technologyLokesh Patil
As consumer awareness of health and wellness rises, the nutraceutical market—which includes goods like functional meals, drinks, and dietary supplements that provide health advantages beyond basic nutrition—is growing significantly. As healthcare expenses rise, the population ages, and people want natural and preventative health solutions more and more, this industry is increasing quickly. Further driving market expansion are product formulation innovations and the use of cutting-edge technology for customized nutrition. With its worldwide reach, the nutraceutical industry is expected to keep growing and provide significant chances for research and investment in a number of categories, including vitamins, minerals, probiotics, and herbal supplements.