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).
A large amount of data is available on the functional impact of missense mutations in TP53 tumor suppressor gene. and on mutation patterns in many different cancers. TP53 direct sequencing is a time-consuming method with limitations in detection level. Here we describe the development and verification of a TP53 Next Generation Sequencing method based on Ion AmpliSeq technology. Preliminary data demonstrate that the TP53 Ion AmpliSeq panel and Ion Reporter Software analysis solution meets the requirements of clinical research laboratories.
Circulating cell free DNA is a potential tumor marker in a non-invasive blood test for the treatment and evaluation of cancer and recurrence monitoring. As circulating tumor DNA is often present at low frequencies within circulating cell free DNA, targeted sequencing on the Ion Torrent™ platform is an optimal tool or mutation detection with very little sample input required. Here, we demonstrate a complete workflow from isolation through molecular characterization of circulating tumor DNA. We have optimized a protocol using magnetic beads to isolate circulating cell free DNA. This protocol is easily automated to process up to 192 samples a day. It is also easily scalable for any input volume and can elute in volumes down to 15 μL resulting in no loss of low frequency alleles. We demonstrate comparable performance between this bead based isolation and column based isolation. We have completed molecular characterization of circulating cell free DNA using the multiplexing capabilities of AmpliSeq™ and the Ion PGM™. With the Ion AmpliSeq™ Cancer Hotspot Panel v2, we performed targeted sequencing of 50 genes of interest, covering 2800 COSMIC mutations. We demonstrate good reproducibility of amplicon representation as well as allelic frequencies. Through saturation studies and subsampling, we have determined the limit of detection of hotspots circulating cell free DNA on the Ion PGM™ to be below 1%. We further demonstrate proof of principle of this workflow on circulating cell free DNA and matched FFPE samples. Our results verify the accuracy and ease of our workflow. This protocol, from isolation through targeted sequencing, will not only result in a simple sample preparation for circulating cell free DNA but also facilitate rapid mutation detection to advance cancer research.
Direct Sanger CE Sequencing of Individual Ampliseq Cancer Panel Targets from ...Thermo Fisher Scientific
The introduction of defined Ion AmpliSeq™ panels for detection and characterization of actionable mutations occurring in tumor tissue has the potential to revolutionize translational oncology research. The Ion Ampliseq™ cancer hot spot panel version 2 (CHP v2) by Ion Torrent includes 207 actionable sequences from a single target and mutation targets present in 50 genes and the more comprehensive Ion Oncomine™ cancer panel (OCP) developed by Life Technologies Compendia Bioscience™ contains over 2000 mutations. A hallmark of these Ion Torrent Ampliseq cancer panels is the low amount of input DNA needed which is critical when the clinical specimen material is limited such as with fine needle biopsy or FFPE samples. Typically, 10 ng of DNA obtained from these sources is sufficient to produce informative sequencing data. Often, cancer-causing or promoting mutations are detected at relatively low allele frequencies like 10-20 % compared to the major normal allele. Many researchers wish to verify these findings of low frequency mutations by an orthologous method such as traditional dye-fluorescent Sanger sequencing on a capillary electrophoresis (CE) instrument such as the Applied Biosystems 3500 genetic analyzer. To that end, we have developed a workflow that enables the amplification and traditional Sanger sequencing of individual Ion AmpliSeq targets directly from the AmpliSeq library starting material.
The method requires a retainer of 1 μl (~ 5%) of the original AmpliSeq preamplification material. A dilution of this aliquot is used as template source for individualized PCR/sequencing reactions. We show that a random selection of 48 targets from the CHPv2 panel could be successfully amplified and Sanger-sequenced from an Ion Torrent Ampliseq library originally prepared from 10 ng of FFPE
DNA. Furthermore, we show the successful Sanger-re-sequencing of all individual 24 targets covering the TP53 exons from the same sample processed and pre-amplified with the OncoMine AmpliSeq panel.
Taken together, this method will enable researchers to reflex-test potential mutations of interest from very material-limited specimen using Sanger CE sequencing
A large amount of data is available on the functional impact of missense mutations in TP53 tumor suppressor gene. and on mutation patterns in many different cancers. TP53 direct sequencing is a time-consuming method with limitations in detection level. Here we describe the development and verification of a TP53 Next Generation Sequencing method based on Ion AmpliSeq technology. Preliminary data demonstrate that the TP53 Ion AmpliSeq panel and Ion Reporter Software analysis solution meets the requirements of clinical research laboratories.
Circulating cell free DNA is a potential tumor marker in a non-invasive blood test for the treatment and evaluation of cancer and recurrence monitoring. As circulating tumor DNA is often present at low frequencies within circulating cell free DNA, targeted sequencing on the Ion Torrent™ platform is an optimal tool or mutation detection with very little sample input required. Here, we demonstrate a complete workflow from isolation through molecular characterization of circulating tumor DNA. We have optimized a protocol using magnetic beads to isolate circulating cell free DNA. This protocol is easily automated to process up to 192 samples a day. It is also easily scalable for any input volume and can elute in volumes down to 15 μL resulting in no loss of low frequency alleles. We demonstrate comparable performance between this bead based isolation and column based isolation. We have completed molecular characterization of circulating cell free DNA using the multiplexing capabilities of AmpliSeq™ and the Ion PGM™. With the Ion AmpliSeq™ Cancer Hotspot Panel v2, we performed targeted sequencing of 50 genes of interest, covering 2800 COSMIC mutations. We demonstrate good reproducibility of amplicon representation as well as allelic frequencies. Through saturation studies and subsampling, we have determined the limit of detection of hotspots circulating cell free DNA on the Ion PGM™ to be below 1%. We further demonstrate proof of principle of this workflow on circulating cell free DNA and matched FFPE samples. Our results verify the accuracy and ease of our workflow. This protocol, from isolation through targeted sequencing, will not only result in a simple sample preparation for circulating cell free DNA but also facilitate rapid mutation detection to advance cancer research.
Direct Sanger CE Sequencing of Individual Ampliseq Cancer Panel Targets from ...Thermo Fisher Scientific
The introduction of defined Ion AmpliSeq™ panels for detection and characterization of actionable mutations occurring in tumor tissue has the potential to revolutionize translational oncology research. The Ion Ampliseq™ cancer hot spot panel version 2 (CHP v2) by Ion Torrent includes 207 actionable sequences from a single target and mutation targets present in 50 genes and the more comprehensive Ion Oncomine™ cancer panel (OCP) developed by Life Technologies Compendia Bioscience™ contains over 2000 mutations. A hallmark of these Ion Torrent Ampliseq cancer panels is the low amount of input DNA needed which is critical when the clinical specimen material is limited such as with fine needle biopsy or FFPE samples. Typically, 10 ng of DNA obtained from these sources is sufficient to produce informative sequencing data. Often, cancer-causing or promoting mutations are detected at relatively low allele frequencies like 10-20 % compared to the major normal allele. Many researchers wish to verify these findings of low frequency mutations by an orthologous method such as traditional dye-fluorescent Sanger sequencing on a capillary electrophoresis (CE) instrument such as the Applied Biosystems 3500 genetic analyzer. To that end, we have developed a workflow that enables the amplification and traditional Sanger sequencing of individual Ion AmpliSeq targets directly from the AmpliSeq library starting material.
The method requires a retainer of 1 μl (~ 5%) of the original AmpliSeq preamplification material. A dilution of this aliquot is used as template source for individualized PCR/sequencing reactions. We show that a random selection of 48 targets from the CHPv2 panel could be successfully amplified and Sanger-sequenced from an Ion Torrent Ampliseq library originally prepared from 10 ng of FFPE
DNA. Furthermore, we show the successful Sanger-re-sequencing of all individual 24 targets covering the TP53 exons from the same sample processed and pre-amplified with the OncoMine AmpliSeq panel.
Taken together, this method will enable researchers to reflex-test potential mutations of interest from very material-limited specimen using Sanger CE sequencing
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.
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.
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 Presence and Persistence of Resistant and Stem Cell-Like Tumor Cells as a...QIAGEN
Epithelial ovarian cancer is the fifth leading cause of cancer-related deaths of women in the United States and Europe and ranks as the second most common type of gynecological malignancy. Most cases are diagnosed in advanced stages and although the response rates to platinum-based chemotherapy are high, the majority of patients nevertheless have poor survival rates. Although the reasons for these poor outcomes are likely to be multifactorial, one particular area of interest has recently focused on hematogenous tumor cell dissemination that has been shown to originate from disseminated tumor cells (DTCs) in the bone marrow (BM) and circulating tumor cells (CTCs) in the blood. Here, we demonstrate that the negative prognostic impact of CTCs and DTCs arise from specific cellular phenotypes and are associated with platinum-resistance and stem cell-associated proteins.
Insights into the tumor microenvironment and therapeutic T cell manufacture r...Thermo Fisher Scientific
TCRβ immune repertoire analysis by next-generation sequencing is emerging as a valuable tool for research studies of the tumor microenvironment and potential immune responses to cancer immunotherapy1-4. Here we describe a multiplex PCR-based TCRβ sequencing assay (Ion AmpliSeqTM Immune Repertoire Assay Plus – TCRβ) that leverages Ion AmpliSeq library construction chemistry and the long read capability of the Ion S5 530TM chip to provide coverage of all three CDR domains of the human TCRβ chain. We demonstrate use of the assay to evaluate tumor-infiltrating T cell repertoire features and monitor manufacture of therapeutic T cells.
A Next-Generation Sequencing Assay to Estimate Tumor Mutation Load at > 5% Al...Thermo Fisher Scientific
Immunotherapies have shown anti-cancer effects in melanoma, NSCLC, and bladder cancer. High tumor mutation load is associated with positive responses from immune checkpoint inhibitors. However, current methods to estimate tumor mutation load often have high infrastructure needs, and require large amounts of DNA.
Sequencing 60,000 Samples: An Innovative Large Cohort Study for Breast Cancer...QIAGEN
This slidedeck focuses on the design of a large cohort study for assessing breast cancer risk and how an innovative digital sequencing approach is able to solve the previously unmet challenges of this type of NGS study design. Our speaker, Dr. Fergus J. Couch of the Mayo Clinic, presents on the design of this NCI-funded project, which comprises the sequencing of 60,000 samples to assess the risk of breast cancer through association with targeted genes. The design and size of the study requires an accurate, robust and high-throughput sequencing method. The investigators are using a digital DNA sequencing approach from QIAGEN that incorporates molecular barcodes to tag and remove PCR duplicates and increase NGS assay sensitivity. The approach also uses proprietary chemistry that enables uniform sequencing to efficiently utilize sequencing power and deliver optimized results.
Proteogenomic analysis of human colon cancer reveals new therapeutic opportun...Gul Muneer
We performed the first proteogenomic study on a prospectively collected colon cancer cohort. Comparative proteomic and phosphoproteomic analysis of paired tumor and normal adjacent tissues produced a catalog of colon cancer-associated proteins and phosphosites, including known and putative new biomarkers, drug targets, and cancer/testis antigens. Proteogenomic integration not only prioritized genomically inferred targets, such as copy-number drivers and mutation-derived neoantigens, but also yielded novel findings. Phosphoproteomics data associated Rb phosphorylation with increased proliferation and decreased apoptosis in colon cancer, which explains why this classical tumor suppressor is amplified in colon tumors and suggests a rationale for targeting Rb phosphorylation in colon cancer. Proteomics identified an association between decreased CD8 T cell infiltration and increased glycolysis in microsatellite instability-high (MSI-H) tumors, suggesting glycolysis as a potential target to overcome the resistance of MSI-H tumors to immune checkpoint blockade. Proteogenomics presents new avenues for biological discoveries and therapeutic development.
Fully automated Ion AmpliSeq™ library preparation using the Ion Chef SystemThermo Fisher Scientific
As next generation sequencing
evolves, the ability to create reproducible, high
quality libraries becomes increasingly
important. The Ion AmpliSeq™ method has
proven useful to reliably generate quality libraries
from as little as 10 ng of nucleic acid. We
developed the Ion Chef™ system to offer an
automated solution for templating and sequencing
chip preparation. Here we demonstrate the
marriage of Ion AmpliSeq reagents with the Ion
Chef system to create a fully automated library
preparation solution. This system includes a new
set of consumables and software to enable DNA to
loaded chip in less than 24 hours with only two
touch points. Eight Ion AmpliSeq libraries can be
prepared in less than 8 hours with less than 15
minutes of hands on time.
Application Note: A Simple One-Step Library Prep Method To Enable AmpliSeq Pa...QIAGEN
Targeted amplicon sequencing is a cost-effective, convenient and rapid method for variant detection. This application note outlines a straightforward workflow that uses the QIAseq 1-Step Amplicon Library kit to verify AmpliSeq targeted sequencing assays on the Illumina sequencing instruments. By combining end-repair and ligation, the QIAseq 1-Step Amplicon Library Kit offers a fast and efficient 30-minute procedure for the preparation of high-quality, artifact-free Illumina libraries from any PCR amplicons, including AmpliSeq Panels.
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.
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.
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 Presence and Persistence of Resistant and Stem Cell-Like Tumor Cells as a...QIAGEN
Epithelial ovarian cancer is the fifth leading cause of cancer-related deaths of women in the United States and Europe and ranks as the second most common type of gynecological malignancy. Most cases are diagnosed in advanced stages and although the response rates to platinum-based chemotherapy are high, the majority of patients nevertheless have poor survival rates. Although the reasons for these poor outcomes are likely to be multifactorial, one particular area of interest has recently focused on hematogenous tumor cell dissemination that has been shown to originate from disseminated tumor cells (DTCs) in the bone marrow (BM) and circulating tumor cells (CTCs) in the blood. Here, we demonstrate that the negative prognostic impact of CTCs and DTCs arise from specific cellular phenotypes and are associated with platinum-resistance and stem cell-associated proteins.
Insights into the tumor microenvironment and therapeutic T cell manufacture r...Thermo Fisher Scientific
TCRβ immune repertoire analysis by next-generation sequencing is emerging as a valuable tool for research studies of the tumor microenvironment and potential immune responses to cancer immunotherapy1-4. Here we describe a multiplex PCR-based TCRβ sequencing assay (Ion AmpliSeqTM Immune Repertoire Assay Plus – TCRβ) that leverages Ion AmpliSeq library construction chemistry and the long read capability of the Ion S5 530TM chip to provide coverage of all three CDR domains of the human TCRβ chain. We demonstrate use of the assay to evaluate tumor-infiltrating T cell repertoire features and monitor manufacture of therapeutic T cells.
A Next-Generation Sequencing Assay to Estimate Tumor Mutation Load at > 5% Al...Thermo Fisher Scientific
Immunotherapies have shown anti-cancer effects in melanoma, NSCLC, and bladder cancer. High tumor mutation load is associated with positive responses from immune checkpoint inhibitors. However, current methods to estimate tumor mutation load often have high infrastructure needs, and require large amounts of DNA.
Sequencing 60,000 Samples: An Innovative Large Cohort Study for Breast Cancer...QIAGEN
This slidedeck focuses on the design of a large cohort study for assessing breast cancer risk and how an innovative digital sequencing approach is able to solve the previously unmet challenges of this type of NGS study design. Our speaker, Dr. Fergus J. Couch of the Mayo Clinic, presents on the design of this NCI-funded project, which comprises the sequencing of 60,000 samples to assess the risk of breast cancer through association with targeted genes. The design and size of the study requires an accurate, robust and high-throughput sequencing method. The investigators are using a digital DNA sequencing approach from QIAGEN that incorporates molecular barcodes to tag and remove PCR duplicates and increase NGS assay sensitivity. The approach also uses proprietary chemistry that enables uniform sequencing to efficiently utilize sequencing power and deliver optimized results.
Proteogenomic analysis of human colon cancer reveals new therapeutic opportun...Gul Muneer
We performed the first proteogenomic study on a prospectively collected colon cancer cohort. Comparative proteomic and phosphoproteomic analysis of paired tumor and normal adjacent tissues produced a catalog of colon cancer-associated proteins and phosphosites, including known and putative new biomarkers, drug targets, and cancer/testis antigens. Proteogenomic integration not only prioritized genomically inferred targets, such as copy-number drivers and mutation-derived neoantigens, but also yielded novel findings. Phosphoproteomics data associated Rb phosphorylation with increased proliferation and decreased apoptosis in colon cancer, which explains why this classical tumor suppressor is amplified in colon tumors and suggests a rationale for targeting Rb phosphorylation in colon cancer. Proteomics identified an association between decreased CD8 T cell infiltration and increased glycolysis in microsatellite instability-high (MSI-H) tumors, suggesting glycolysis as a potential target to overcome the resistance of MSI-H tumors to immune checkpoint blockade. Proteogenomics presents new avenues for biological discoveries and therapeutic development.
Fully automated Ion AmpliSeq™ library preparation using the Ion Chef SystemThermo Fisher Scientific
As next generation sequencing
evolves, the ability to create reproducible, high
quality libraries becomes increasingly
important. The Ion AmpliSeq™ method has
proven useful to reliably generate quality libraries
from as little as 10 ng of nucleic acid. We
developed the Ion Chef™ system to offer an
automated solution for templating and sequencing
chip preparation. Here we demonstrate the
marriage of Ion AmpliSeq reagents with the Ion
Chef system to create a fully automated library
preparation solution. This system includes a new
set of consumables and software to enable DNA to
loaded chip in less than 24 hours with only two
touch points. Eight Ion AmpliSeq libraries can be
prepared in less than 8 hours with less than 15
minutes of hands on time.
Application Note: A Simple One-Step Library Prep Method To Enable AmpliSeq Pa...QIAGEN
Targeted amplicon sequencing is a cost-effective, convenient and rapid method for variant detection. This application note outlines a straightforward workflow that uses the QIAseq 1-Step Amplicon Library kit to verify AmpliSeq targeted sequencing assays on the Illumina sequencing instruments. By combining end-repair and ligation, the QIAseq 1-Step Amplicon Library Kit offers a fast and efficient 30-minute procedure for the preparation of high-quality, artifact-free Illumina libraries from any PCR amplicons, including AmpliSeq Panels.
Cytochrome P450 enzymes metabolize about 75% of drugs, including oncology drugs, with UGT enzymes metabolizing about another 15%. Variations in gene sequence or in copy number may result in an inactive, defective, unstable, mis-spliced, low expressed, or absent enzyme, an increase in enzyme activity, or an altered affinity for substrates. Pharmacogenomics genes can predict whether an individual is a poor or rapid metabolizer, facilitating dose optimization. Failure to adjust dosage of drugs metabolized by the relevant enzyme can lead to adverse drug reaction, or conversely to too rapid drug metabolism and no drug response. Here, we present a pharmacogenomics (PGx) Research panel to detect 139 SNV/Indel targets in 42 genes (Figure 1) and CYP2D6 copy number variation (CNV, Figure 2). This panel covers the commonly known targets in genes encoding drug metabolism enzymes and associated transport proteins. The panel design is particularly challenging due to high levels of sequence homology between the cytochrome P450 genes. This assay uses Ion AmpliSeq™ technology and contains 146 amplicons in an ultrahigh multiplex PCR in a single pool, followed by Ion Torrent™ semiconductor sequencing. The assay requires as little as 10 ng of input DNA. This customizable panel allows target addition or removal, and will be the first NGS PGx Research panel on the market.
Improved Reagents & Methods for Target Enrichment in Next Generation Sequencing, presented by Dr Mark Behlke, Chief Scientific Officer at Integrated DNA Technologies
Cancer therapies that target specific pathways can be more effective than established, nonspecific chemotherapy and radiation treatments, and may prevent side effects on healthy tissues. Such targeted therapies can only be applied after underlying gene mutations have been identified. However, detecting low frequency variants from clinically relevant samples poses significant challenges. Specimens are routinely formalin-fixed and paraffin-embedded (FFPE) for histology, which can decrease the efficiency of NGS library preparation. In this presentation, we discuss approaches for extraction of DNA from FFPE samples, and recommend quality control assays to guide parameter selection for library construction and sequencing depth.
Struggling with low editing efficiency or delivery problems in primary or difficult-to-transfect cells? In this presentation, learn about the advantages of using a Cas9:crRNA:tracrRNA ribonucleoprotein (RNP) complex for genome editing. We show the benefits of using RNP complexes, including ease of use, limiting off-target effects, and stability. We also present data showing how genome editing efficiency rates are improved by our Cas9 electroporation enhancer. Furthermore, we provide advice on how to optimize transfection using the Alt-R™ CRISPR-Cas9 System in combination with different electroporation methodologies.
(December 2, 2021) The Bench to Bedside Series: Preclinical Cancer Research w...Scintica Instrumentation
Overview:
The goal of this webinar will be to provide a high-level overview of the various stages of preclinical cancer research and discuss the role that innovative instrumentation can play in moving science forward.
To better understand how to treat and control cancer, researchers start by investigating the basics – the cells, molecules, and genes that make up the human body. This type of study, which is often referred to as basic or discovery research, aims to understand the underlying mechanisms contributing to cancer growth and spread. This knowledge is an essential starting point for developing future diagnostic tests and treatment strategies.
After finding an innovative idea that works in cells, researchers need to take their studies to the next level by employing animal models that have similar biology to humans. Animal models have helped scientists make some of the most important cancer discoveries over the years. Furthermore, preclinical imaging technologies allow researchers to perform longitudinal animal studies that are noninvasive leaving the underlying biology intact so that one can track changes throughout the entire disease process.
It was previously thought that the journey from bench to bedside was unidirectional, starting with discovery research and moving towards clinical trials. However, in the last decade, it has become crucial for basic scientists and clinicians to work together towards finding innovative solutions that will positively impact patient care.
After attending this webinar, we hope you will have a better understanding of the preclinical workflow needed to push an idea from bench to bedside as well as some of the key equipment that is needed along the way.
This webinar series will be hosted by Drs. Katie Parkins and Tyler Lalonde, both of which have extensive experience in translational research areas including oncology, neuroscience, molecular imaging, and drug development.
In this webinar we will discuss the following topics:
• Introduction To Cancer Research
• What does “Bench to Bedside” mean?
• In vitro characterization
• Rapid throughput screening
• Quantitative tools
• Moving towards translation
Next generation sequencing (NGS) of circulating tumor DNA (ctDNA) from patient plasma is becoming more widespread in oncology clinical trials. The noninvasive nature of acquiring these samples is particularly important when resection of representative tumor samples is not advised or not possible. However, profiling of ctDNA has challenges to overcome, such as low concentration of ctDNA shed from the tumor and a low signal:noise ratio caused by somatic alterations with less than 1% variant allele fraction. Improving the sensitivity of these assays to detect low allele frequency events with high confidence requires robust sequencing of low input libraries while employing error correction to reduce background noise. To overcome these challenges, we have incorporated unique molecular identifiers (UMIs) into our NGS workflow. Using these novel adapters paired with our proprietary bioinformatics pipeline (AstraZeneca), the number of false positive variants reported for allele fractions less than 0.5% was reduced tenfold. We also refined our curation based on the mapping quality and strand bias in the vicinity of each variant to further reduce the background noise. The use of xGen® Dual Index UMI Adapters—Tech Access (Integrated DNA Technologies) has enabled us to sequence thousands of plasma samples from diverse tumor indications and at differing time points during our trials. The generated data are highly informative with the potential to answer critical questions relating to individual response or resistance to experimental therapies. During this webinar, we discuss our current NGS ctDNA workflow and our future plans to increase our sequencing sensitivity with these novel UMI adapters.
Estimating Mutation Load from Tumor Research Samples using a Targeted Next-Ge...Thermo Fisher Scientific
Tumor mutation load predicts durable benefit from immune checkpoint inhibitors in several cancer types. Existing methods to estimate tumor mutation load have large input DNA and extensive infrastructure requirements and are associated with delays due to shipping biopsy samples to central laboratories. We demonstrate the ability of a targeted panel with fast
turn-around time and low input requirement for estimating mutation load from tumor samples to advance research in immuno-oncology.
Robert P. Edwards, MD, Chair of OB/GYN/RS, Co-Director of Women's Cancer Program at University of Pittsburgh, offers information about the current state of immunotherapy for recurrent ovarian cancer patients.
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.
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.
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.
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
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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 .
Slide 1: Title Slide
Extrachromosomal Inheritance
Slide 2: Introduction to Extrachromosomal Inheritance
Definition: Extrachromosomal inheritance refers to the transmission of genetic material that is not found within the nucleus.
Key Components: Involves genes located in mitochondria, chloroplasts, and plasmids.
Slide 3: Mitochondrial Inheritance
Mitochondria: Organelles responsible for energy production.
Mitochondrial DNA (mtDNA): Circular DNA molecule found in mitochondria.
Inheritance Pattern: Maternally inherited, meaning it is passed from mothers to all their offspring.
Diseases: Examples include Leber’s hereditary optic neuropathy (LHON) and mitochondrial myopathy.
Slide 4: Chloroplast Inheritance
Chloroplasts: Organelles responsible for photosynthesis in plants.
Chloroplast DNA (cpDNA): Circular DNA molecule found in chloroplasts.
Inheritance Pattern: Often maternally inherited in most plants, but can vary in some species.
Examples: Variegation in plants, where leaf color patterns are determined by chloroplast DNA.
Slide 5: Plasmid Inheritance
Plasmids: Small, circular DNA molecules found in bacteria and some eukaryotes.
Features: Can carry antibiotic resistance genes and can be transferred between cells through processes like conjugation.
Significance: Important in biotechnology for gene cloning and genetic engineering.
Slide 6: Mechanisms of Extrachromosomal Inheritance
Non-Mendelian Patterns: Do not follow Mendel’s laws of inheritance.
Cytoplasmic Segregation: During cell division, organelles like mitochondria and chloroplasts are randomly distributed to daughter cells.
Heteroplasmy: Presence of more than one type of organellar genome within a cell, leading to variation in expression.
Slide 7: Examples of Extrachromosomal Inheritance
Four O’clock Plant (Mirabilis jalapa): Shows variegated leaves due to different cpDNA in leaf cells.
Petite Mutants in Yeast: Result from mutations in mitochondrial DNA affecting respiration.
Slide 8: Importance of Extrachromosomal Inheritance
Evolution: Provides insight into the evolution of eukaryotic cells.
Medicine: Understanding mitochondrial inheritance helps in diagnosing and treating mitochondrial diseases.
Agriculture: Chloroplast inheritance can be used in plant breeding and genetic modification.
Slide 9: Recent Research and Advances
Gene Editing: Techniques like CRISPR-Cas9 are being used to edit mitochondrial and chloroplast DNA.
Therapies: Development of mitochondrial replacement therapy (MRT) for preventing mitochondrial diseases.
Slide 10: Conclusion
Summary: Extrachromosomal inheritance involves the transmission of genetic material outside the nucleus and plays a crucial role in genetics, medicine, and biotechnology.
Future Directions: Continued research and technological advancements hold promise for new treatments and applications.
Slide 11: Questions and Discussion
Invite Audience: Open the floor for any questions or further discussion on the topic.
Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
Monitor common gases, weather parameters, particulates.
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.
Richard's entangled aventures in wonderlandRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
Observation of Io’s Resurfacing via Plume Deposition Using Ground-based Adapt...Sérgio Sacani
Since volcanic activity was first discovered on Io from Voyager images in 1979, changes
on Io’s surface have been monitored from both spacecraft and ground-based telescopes.
Here, we present the highest spatial resolution images of Io ever obtained from a groundbased telescope. These images, acquired by the SHARK-VIS instrument on the Large
Binocular Telescope, show evidence of a major resurfacing event on Io’s trailing hemisphere. When compared to the most recent spacecraft images, the SHARK-VIS images
show that a plume deposit from a powerful eruption at Pillan Patera has covered part
of the long-lived Pele plume deposit. Although this type of resurfacing event may be common on Io, few have been detected due to the rarity of spacecraft visits and the previously low spatial resolution available from Earth-based telescopes. The SHARK-VIS instrument ushers in a new era of high resolution imaging of Io’s surface using adaptive
optics at visible wavelengths.
Assessment of TP53 Mutation Status in Breast Tumor Tissue using the "Ion AmpliseqTM TP53 Panel"
1. Assessment of TP53 Mutation Status in Breast Tumor
Tissue using the "Ion Ampliseq™ TP53 Panel"
Laxmi Silwal-Pandit1,2, Einar Rødland2, Astrid M Dalsgaard1, Inger Riise Bergheim2, Suet-Feung Chin3,4, Carlos Caldas3,4,
Anne-Lise Børresen-Dale1,2 and Anita Langerød1,2
1Department of Genetics, Institute for Cancer Research, Oslo University Hospital Radiumhospitalet, Norway. 2The K.G. Jebsen Center for Breast Cancer Research, Institute for Clinical
Medicine, Faculty of Medicine, University of Oslo, Norway. 3Cancer Research UK, Cambridge Institute, UK. 4Department of Oncology, University of Cambridge, UK.
Aim
The aim was to develop and evaluate a Next Generation
Sequencing method for TP53 mutation analysis covering the huge
spectrum of mutations in the gene, and with a sequencing depth
facilitating the study of clonal and subclonal variants in breast
tumor tissue.
Material & Methods
Study design:
The verification of the panel has been performed in two phases, and
here we present the results of phase 1 and 2A (Fig.1). DNA from fresh
frozen samples (METABRIC cohort) previously characterized by Sanger
sequencing were analyzed1.
Panel design:
Non-overlapping primers (Fig. 2) were designed to provide:
• Full coverage of all coding exons and exon-intron boundaries
• Overlapping amplicons covering all exons
• Minimal off-target sequencing
• No SNPs in the last five nucleotides of primer
• Amplification of DNA extracted from FFPE material (Fig.2)
Sequencing & data analysis:
The sequencing were carried out following standard protocol for the
Ion PGM™ Sequencer. (Fig.3) Data analysis was performed on Ion
Reporter TM Software 4.0 using the TP53 panel workflow.
Results
The new panel covers TP53 coding regions comprehensively. It is
comprised of 24 primer pairs across 2 pools requiring 20 ng of DNA.
The design of short amplicons (73 to 139 bp) permits the amplification
of DNA from FFPE.
Phase 1 were carried out loading 10 samples on an Ion 318™ chip,
giving a high coverage (>500x) in 95% of the sequenced amplicons.
Average coverage uniformity was 88% for all samples and the
percentage of mapped reads on target was 97%. The overall sensitivity
was 95% (Table 1).
In Phase 2A 200 samples were analyzed loading 20 samples per 318™
chip, giving a high coverage (>500x) in 97% of sequenced amplicons.
Average coverage uniformity was 86% for all samples and the
percentage of mapped reads on target was 94%. The overall sensitivity
was 90% (Table 2). Three large deletions were not detected, whereas
two new subclonal base substitutions were found (double mutations).
Conclusion
Most mutations detected by Sanger Sequencing were also
detected by the TP53 panel, but large deletions failed to be
identified. Very few subclonal mutations were seen using ultra-
deep sequencing. The Ion Reporter facilitates data analysis for
clinical research labs with limited bioinformatic resources.
Introduction
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 Torrent™ next-generation semiconductor
sequencing and Ion AmpliSeq™ technology (Life Technologies™).
Figure 3: Ion AmpliSeq™ Workflow
Acknowledgements
We would like to acknowledge Rosella Petraroli , Chrysanthi Ainali, and Alain
Rico at Thermo Fisher Scientific for great collaboration during the project.
Figure 2: Ion AmpliSeq ™ TP53 Panel design
International p53 Workshop, July 15-19, 2014, Stockholm
Reference: 1Silwal-Pandit et al, Clin Cancer Res; 20(13) July 1, 2014.
Figure 1: Study design
Phase 1
Analysis parameter optimization
Reproducibility study
• 30 breast tumor samples
• Analyzed in 2 labs
• Known mutation status
• Selected cohort (20 mut, 10 wt)
Phase 2A
Extended performance verification
• 200 breast tumor samples
• Known mutation status
• Unselected cohort (with regard
to mutation status)
Phase 2B
• Additional 200 tumor samples
Mutation type Mutations (no.) Miscalls (no.) Sensitivity (%)
Base substitution 10a 0 -
Deletion 6 0 -
Insertion 5 1b -
Complex (in+del) 0 0 -
TOTAL 21 8 95%
Mutation type Mutations (no.) Miscalls (no.) Sensitivity (%)
Base substitution 43 2 -
Deletion 12 3ab -
Insertion 3 0 -
Complex (ins+del) 1 1c -
TOTAL 59d 6 90%
Table 1: Sensitivity, phase 1
Table 2: Sensitivity, phase 2A
a18bp deletions close to amplicon end misaligned to reference.
b24bp deletion at amplicon end; no sequence on opposite side to allow calling.
c Complex mutation (ins+del) visible in sequence data, but not called.
d Totally 53 different type of mutations across the gene.
aOne mut previously detected by TTGE, and not by Sanger Seq, was characterized.
bComplex duplication of 18bp.