This presentation provides an overview of performing allele-specific copy number variation (CNV) analysis using digital PCR. We demonstrate a pharmacogenomics analysis workflow using the QuantStudio® 12k Flex system to perform CYP2D6 SNP genotyping and copy number analysis, followed by allele-specific copy number analysis using TaqMan® SNP Genotyping Assays on the QuantStudio® 3D digital PCR system. Digital PCR enables precise determination of the specific allele composition of samples with duplications in the CYP2D6 gene, which can impact the extent to which certain drugs are metabolized.
Non-coding RNAs (ncRNAs) are functional RNA molecules that are not translated into proteins. There are several types of ncRNAs that play important roles in biological processes. tRNAs help translate nucleotides into amino acids during protein synthesis. rRNA and snoRNAs are involved in ribosome and RNA structure/modification. MiRNAs regulate gene expression by binding to mRNA. LncRNAs regulate processes like chromatin structure and transcription. Mt-tRNAs specific to mitochondria are essential for oxidative phosphorylation. Mutations can cause diseases like myopathies.
This document provides an overview of downstream analyses that can be performed after variant identification and filtering in a typical variant calling pipeline. It discusses visualization of variant data in each gene to identify potential causative variants. It also mentions association studies as another type of downstream analysis where variants are tested for association with disease phenotypes. The goal of downstream analyses is to help prioritize variants for further investigation.
This document discusses long non-coding RNAs (lncRNAs). It begins by describing the discovery of lncRNAs in the 1980s-2000s through cDNA sequencing. It then states that lncRNAs are the largest class of transcripts in mouse and human genomes. The document discusses that lncRNAs were once thought to be useless but are now known to have regulatory functions. It provides details on the characteristics, locations in the genome, functions, mechanisms of action, roles in human disease, and implications in human carcinomas of lncRNAs.
Course: Bioinformatics for Biomedical Research (2014).
Session: 4.1- Introduction to RNA-seq and RNA-seq Data Analysis.
Statistics and Bioinformatisc Unit (UEB) & High Technology Unit (UAT) from Vall d'Hebron Research Institute (www.vhir.org), Barcelona.
The document discusses transcriptomics and the relationship between transcriptome size and organism complexity. It questions how gene expression contributes to transcriptome size and what new studies reveal about size and complexity. Specifically, it notes that alternative splicing and RNA editing increase transcriptome size and complexity. It also discusses that the human genome is pervasively transcribed, with one stretch of DNA encoding many RNAs, including microRNAs, which control mRNA expression and are involved in development, gene regulation, and diseases like cancer.
This document provides an overview and introduction to RNA-seq analysis using Next Generation Sequencing. It discusses the RNA-seq workflow including mapping reads with TopHat2, transcript assembly with Cufflinks, and differential expression analysis. Key points covered include the advantages of RNA-seq over microarrays, the exponential drop in sequencing costs, mapping strategies for junction reads including TopHat, and running TopHat from the command line.
1. A DNA microarray contains thousands of DNA probes attached to a solid surface in defined locations. Each probe represents a single gene.
2. Sample mRNA is converted to fluorescently labeled cDNA and hybridized to the DNA microarray. The level of fluorescence indicates the expression level of each gene.
3. After washing, the microarray is scanned and analyzed to determine changes in gene expression between control and test samples. This allows high-throughput analysis of gene expression profiles.
Thousands of different long non-coding RNAs (lncRNAs) exist in mammalian cells. lncRNAs do not encode proteins but can be very important for cell function. Studying their functions can be difficult because of their diverse modes of action. One method to discern cellular function is by selective knockdown of a specific lncRNA species. However, achieving consistent knockdown has proven to be more challenging for lncRNAs than for mRNAs or miRNAs. In this presentation, we discuss some of the issues encountered with lncRNA research. We cover antisense oligonucleotide (ASO) and small interfering RNA (siRNA) methods for lncRNA knockdown. And, we show how cellular localization of a specific lncRNA target informs the choice of knockdown method.
Non-coding RNAs (ncRNAs) are functional RNA molecules that are not translated into proteins. There are several types of ncRNAs that play important roles in biological processes. tRNAs help translate nucleotides into amino acids during protein synthesis. rRNA and snoRNAs are involved in ribosome and RNA structure/modification. MiRNAs regulate gene expression by binding to mRNA. LncRNAs regulate processes like chromatin structure and transcription. Mt-tRNAs specific to mitochondria are essential for oxidative phosphorylation. Mutations can cause diseases like myopathies.
This document provides an overview of downstream analyses that can be performed after variant identification and filtering in a typical variant calling pipeline. It discusses visualization of variant data in each gene to identify potential causative variants. It also mentions association studies as another type of downstream analysis where variants are tested for association with disease phenotypes. The goal of downstream analyses is to help prioritize variants for further investigation.
This document discusses long non-coding RNAs (lncRNAs). It begins by describing the discovery of lncRNAs in the 1980s-2000s through cDNA sequencing. It then states that lncRNAs are the largest class of transcripts in mouse and human genomes. The document discusses that lncRNAs were once thought to be useless but are now known to have regulatory functions. It provides details on the characteristics, locations in the genome, functions, mechanisms of action, roles in human disease, and implications in human carcinomas of lncRNAs.
Course: Bioinformatics for Biomedical Research (2014).
Session: 4.1- Introduction to RNA-seq and RNA-seq Data Analysis.
Statistics and Bioinformatisc Unit (UEB) & High Technology Unit (UAT) from Vall d'Hebron Research Institute (www.vhir.org), Barcelona.
The document discusses transcriptomics and the relationship between transcriptome size and organism complexity. It questions how gene expression contributes to transcriptome size and what new studies reveal about size and complexity. Specifically, it notes that alternative splicing and RNA editing increase transcriptome size and complexity. It also discusses that the human genome is pervasively transcribed, with one stretch of DNA encoding many RNAs, including microRNAs, which control mRNA expression and are involved in development, gene regulation, and diseases like cancer.
This document provides an overview and introduction to RNA-seq analysis using Next Generation Sequencing. It discusses the RNA-seq workflow including mapping reads with TopHat2, transcript assembly with Cufflinks, and differential expression analysis. Key points covered include the advantages of RNA-seq over microarrays, the exponential drop in sequencing costs, mapping strategies for junction reads including TopHat, and running TopHat from the command line.
1. A DNA microarray contains thousands of DNA probes attached to a solid surface in defined locations. Each probe represents a single gene.
2. Sample mRNA is converted to fluorescently labeled cDNA and hybridized to the DNA microarray. The level of fluorescence indicates the expression level of each gene.
3. After washing, the microarray is scanned and analyzed to determine changes in gene expression between control and test samples. This allows high-throughput analysis of gene expression profiles.
Thousands of different long non-coding RNAs (lncRNAs) exist in mammalian cells. lncRNAs do not encode proteins but can be very important for cell function. Studying their functions can be difficult because of their diverse modes of action. One method to discern cellular function is by selective knockdown of a specific lncRNA species. However, achieving consistent knockdown has proven to be more challenging for lncRNAs than for mRNAs or miRNAs. In this presentation, we discuss some of the issues encountered with lncRNA research. We cover antisense oligonucleotide (ASO) and small interfering RNA (siRNA) methods for lncRNA knockdown. And, we show how cellular localization of a specific lncRNA target informs the choice of knockdown method.
The document summarizes Ion Torrent next generation sequencing. It detects chemiluminescence given off when a proton is released during sequencing. DNA fragments are attached to beads and undergo emulsion PCR amplification. Sequencing is based on standard pyrosequencing and measures light from chemiluminescent reagents released during the reaction. The output is in FASTQ format.
Kashikant Yadav presented on siRNA (short interfering RNA). siRNA is 20-25 base pairs long, similar to miRNA, and operates in the RNA interference pathway by degrading mRNA with complementary sequences, preventing translation. There are three main methods of siRNA synthesis: chemical synthesis, in vitro transcription, and digestion of long dsRNA by RNAase III or Dicer. siRNA has significance for protecting against viruses, maintaining genome stability, and offers a new tool to specifically repress genes. Potential applications include testing gene function, target validation, pathway analysis, and developing siRNA therapeutics.
This document discusses PIWI-interacting RNAs (piRNAs), a class of small non-coding RNAs that interact with PIWI proteins. It describes how piRNAs were discovered in Drosophila and their role in silencing transposons in the germline. The document outlines piRNA biogenesis, including their location in clusters in genomes and the "ping-pong" mechanism of biogenesis. It also discusses compartmentalization of the piRNA pathway and functions of piRNAs in maintaining genome integrity, transposon silencing, and fertility.
The document summarizes Ion Torrent sequencing technology. It detects hydrogen ions released during DNA polymerization rather than using optics. The sequencing occurs on semiconductor chips patterned through photolithography into wells, each sequencing a different template. As nucleotides are incorporated, hydrogen ions change the pH detected by ion sensors below each well. This allows massively parallel sequencing that is faster, cheaper and simpler than previous technologies.
The document provides an overview of the history and techniques of transcriptome analysis. It discusses how RNA was separated from DNA with the formulation of the central dogma in 1958. Key developments include the discoveries of messenger RNA, transfer RNA, and ribosomal RNA in the 1960s. The document outlines techniques such as serial analysis of gene expression (SAGE) and RNA sequencing (RNA-seq) that allow comprehensive analysis of gene expression patterns. It provides details on the basic steps and advantages of SAGE and describes how next generation sequencing revolutionized transcriptome analysis through massive parallel sequencing.
Protein microarrays, ICAT, and HPLC protein purificationRaul Soto
The document discusses the Isotope-Coded Affinity Tag (ICAT) method for protein quantification and identification. ICAT uses chemical labeling reagents that specifically label cysteine residues. There are 4 main steps: 1) Lyse and label protein samples from two states with light and heavy ICAT tags, 2) Mix and proteolyze samples to generate peptide fragments, some tagged, 3) Isolate tagged fragments using avidin affinity chromatography, 4) Analyze isolated peptides using mass spectrometry to identify and quantify proteins between the two states. ICAT allows accurate quantification of complex protein mixtures.
Whole genome sequencing analysis involves aligning sequencing reads, calling variants like SNPs, indels and structural variants, and annotating the variants. The pipeline includes alignment with tools like BWA, variant calling with GATK for SNPs/indels and Lumpy/Manta for structural variants, and annotation with Ensembl VEP. WGS can detect more types of variants than exome sequencing and has increasing diagnostic utility for diseases.
This document discusses microRNAs (miRNAs), including their biogenesis and function. It covers that miRNAs are small noncoding RNAs around 22 nucleotides long that are found in plants, animals and viruses and function in RNA silencing. It then summarizes the key steps in miRNA biogenesis, from transcription of miRNA genes by RNA polymerase II, to nuclear and cytoplasmic processing by the enzymes Drosha, Exportin 5, Dicer and Argonaute to produce the mature miRNA loaded into the RISC complex.
Accurate DNA Methylation Analysis with Successful Bisulfite Conversion WebinarQIAGEN
Bisulfite conversion is a popular used method for DNA methylation analysis. It is the most convenient and effective way to map DNA methylation to individual bases. The efficiency of the bisulfite conversion has a huge impact on the reliability of the downstream analysis methods and complete conversion is a prerequisite for correct determination of methylation. However, standard methods require harsh conditions with long incubations times at high temperatures and low pH to achieve complete conversion. This harsh treatment can lead to DNA degradation, lowering the yields and sensitivity of the subsequent analysis.
This slidedeck:
• Explains the principle of bisulfite conversion
• Points out the challenges and critical factors for successful bisulfite conversion
• Describes how to overcome the challenges with QIAGEN’s EpiTect Fast Bisulfite Kits
• Gives general recommendations for planning successful bisulfite conversion experiments
MicroRNA and thier role in gene regulationIbad khan
MicroRNAs are small non-coding RNAs that regulate gene expression post-transcriptionally. They were first discovered in 1993 and their biogenesis involves two key steps - processing in the nucleus by the Drosha-DGCR8 complex into pre-miRNAs, followed by export to the cytoplasm and further processing by the Dicer enzyme into mature miRNA. The miRNA is then loaded into the RISC complex containing Argonaute proteins and guides it to target mRNAs to repress translation or promote degradation. MicroRNAs play important roles in various cellular functions and diseases by mediating gene silencing through nine different mechanisms.
Real Time PCR allows for detection and quantification of DNA as amplification occurs. It monitors fluorescence at each cycle to measure DNA accumulation. There are two main types of instrumentation - two-step qRT-PCR which involves reverse transcription followed by PCR, and one-step which combines these steps. Detection relies on fluorescent dyes like SYBR Green or target-specific Taqman probes. Real Time PCR provides advantages over conventional PCR like not requiring gels and being faster and less complex for quantification.
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).
Northern blotting is a technique used to detect specific RNA sequences in a sample. It involves isolating RNA, separating it via gel electrophoresis, transferring it to a membrane, then hybridizing probes with complementary sequences to the target RNA. This allows visualization of gene expression patterns between tissues, developmental stages, and disease states from the detected RNA sequences.
This document provides information about zinc finger proteins. It begins with an introduction to zinc finger motifs, which are protein structural domains characterized by the coordination of zinc ions. The document then discusses the history of zinc finger discovery, functions, and families. It provides details on the most common Cys2His2 zinc finger proteins and their role in DNA recognition and transcriptional regulation. The document also examines uses of zinc finger nucleases for genome editing and their mechanism of action involving creating double-strand breaks in DNA.
Gap-PCR is a PCR technique used to detect deletions or mutations between two known sequences. It uses primer pairs that flank the suspected deletion region to generate products of different sizes depending on whether the deletion is present or not. For example, a normal allele would generate a shorter product than a mutant allele containing the deletion. This allows gap-PCR to efficiently detect deletions larger than 2kb and identify individuals that are heterozygous for the deletion. It has applications in detecting gene deletions associated with conditions like thalassemia.
RNAi – Mechanism and Its Application In Crop Improvementkundan Jadhao
This document summarizes an RNAi presentation on crop improvement using RNA interference. The 3-sentence summary is:
RNA interference (RNAi) is a process of post-transcriptional gene silencing mediated by small RNA molecules. The presentation described the RNAi pathway and various applications of RNAi technology in crop improvement, including increasing nutrient levels, developing virus and pest resistance, and reducing anti-nutritional compounds. Several case studies were provided that demonstrated how RNAi has been used to successfully modify traits in different crops like maize, cotton, coffee, and banana.
Protein-DNA interactions can be either specific or non-specific. Specific interactions involve transcription factors that regulate gene expression by binding to DNA motifs through domains like helix-loop-helix, leucine zipper, or zinc finger motifs. Non-specific interactions involve histones that help structure DNA into nucleosomes within chromatin and can be chemically modified through methylation, demethylation, acetylation, and phosphorylation.
The document summarizes various unsupervised learning algorithms used for analyzing gene expression data from microarray experiments, including k-means clustering, self-organizing maps, and hierarchical clustering. It describes how these algorithms group genes based on similarity in their expression profiles across different conditions or cell types without external labels, helping to simplify data sets and identify genes that may be co-regulated or serve similar functions.
Tamoxifen And CYP2D6: Using Pharmacogenetics to discover a new drugRyan Squire
Dr. Matthew Goetz, assistant professor of oncology and pharmacology at the Mayo Clinic, shared his pharmacogenomic research findings related to risks and occurrence of breast cancer. He explained that in order to truly personalize medicine, you must account for all possible theories and variables. Goetz continued to say that although many believe pharmacology to be boring, it is a key component of the future model of care. Some may say, so this drug doesn’t work–why not just try another drug? It’s much more complicated than that.
Dr. Goetz touched on the variety of cases in his study in breast cancer patients, some with strange and perplexing results. When giving the same drug to multiple patients, each yielded a variety of different results. Some patients had successful reduction in tumor size, while others resulted in no change and some even experienced tumor growth as a result of the drug. Personalized health care is the answer to this, for lack of a better term, ’shot-in-the-dark’ type of therapy. If physicians can understand each patient’s biology and genetic makeup individually, they can better apply treatments and medications. This would therefore reduce health care costs and enable patients to receive much more efficient treatments.
The document summarizes Ion Torrent next generation sequencing. It detects chemiluminescence given off when a proton is released during sequencing. DNA fragments are attached to beads and undergo emulsion PCR amplification. Sequencing is based on standard pyrosequencing and measures light from chemiluminescent reagents released during the reaction. The output is in FASTQ format.
Kashikant Yadav presented on siRNA (short interfering RNA). siRNA is 20-25 base pairs long, similar to miRNA, and operates in the RNA interference pathway by degrading mRNA with complementary sequences, preventing translation. There are three main methods of siRNA synthesis: chemical synthesis, in vitro transcription, and digestion of long dsRNA by RNAase III or Dicer. siRNA has significance for protecting against viruses, maintaining genome stability, and offers a new tool to specifically repress genes. Potential applications include testing gene function, target validation, pathway analysis, and developing siRNA therapeutics.
This document discusses PIWI-interacting RNAs (piRNAs), a class of small non-coding RNAs that interact with PIWI proteins. It describes how piRNAs were discovered in Drosophila and their role in silencing transposons in the germline. The document outlines piRNA biogenesis, including their location in clusters in genomes and the "ping-pong" mechanism of biogenesis. It also discusses compartmentalization of the piRNA pathway and functions of piRNAs in maintaining genome integrity, transposon silencing, and fertility.
The document summarizes Ion Torrent sequencing technology. It detects hydrogen ions released during DNA polymerization rather than using optics. The sequencing occurs on semiconductor chips patterned through photolithography into wells, each sequencing a different template. As nucleotides are incorporated, hydrogen ions change the pH detected by ion sensors below each well. This allows massively parallel sequencing that is faster, cheaper and simpler than previous technologies.
The document provides an overview of the history and techniques of transcriptome analysis. It discusses how RNA was separated from DNA with the formulation of the central dogma in 1958. Key developments include the discoveries of messenger RNA, transfer RNA, and ribosomal RNA in the 1960s. The document outlines techniques such as serial analysis of gene expression (SAGE) and RNA sequencing (RNA-seq) that allow comprehensive analysis of gene expression patterns. It provides details on the basic steps and advantages of SAGE and describes how next generation sequencing revolutionized transcriptome analysis through massive parallel sequencing.
Protein microarrays, ICAT, and HPLC protein purificationRaul Soto
The document discusses the Isotope-Coded Affinity Tag (ICAT) method for protein quantification and identification. ICAT uses chemical labeling reagents that specifically label cysteine residues. There are 4 main steps: 1) Lyse and label protein samples from two states with light and heavy ICAT tags, 2) Mix and proteolyze samples to generate peptide fragments, some tagged, 3) Isolate tagged fragments using avidin affinity chromatography, 4) Analyze isolated peptides using mass spectrometry to identify and quantify proteins between the two states. ICAT allows accurate quantification of complex protein mixtures.
Whole genome sequencing analysis involves aligning sequencing reads, calling variants like SNPs, indels and structural variants, and annotating the variants. The pipeline includes alignment with tools like BWA, variant calling with GATK for SNPs/indels and Lumpy/Manta for structural variants, and annotation with Ensembl VEP. WGS can detect more types of variants than exome sequencing and has increasing diagnostic utility for diseases.
This document discusses microRNAs (miRNAs), including their biogenesis and function. It covers that miRNAs are small noncoding RNAs around 22 nucleotides long that are found in plants, animals and viruses and function in RNA silencing. It then summarizes the key steps in miRNA biogenesis, from transcription of miRNA genes by RNA polymerase II, to nuclear and cytoplasmic processing by the enzymes Drosha, Exportin 5, Dicer and Argonaute to produce the mature miRNA loaded into the RISC complex.
Accurate DNA Methylation Analysis with Successful Bisulfite Conversion WebinarQIAGEN
Bisulfite conversion is a popular used method for DNA methylation analysis. It is the most convenient and effective way to map DNA methylation to individual bases. The efficiency of the bisulfite conversion has a huge impact on the reliability of the downstream analysis methods and complete conversion is a prerequisite for correct determination of methylation. However, standard methods require harsh conditions with long incubations times at high temperatures and low pH to achieve complete conversion. This harsh treatment can lead to DNA degradation, lowering the yields and sensitivity of the subsequent analysis.
This slidedeck:
• Explains the principle of bisulfite conversion
• Points out the challenges and critical factors for successful bisulfite conversion
• Describes how to overcome the challenges with QIAGEN’s EpiTect Fast Bisulfite Kits
• Gives general recommendations for planning successful bisulfite conversion experiments
MicroRNA and thier role in gene regulationIbad khan
MicroRNAs are small non-coding RNAs that regulate gene expression post-transcriptionally. They were first discovered in 1993 and their biogenesis involves two key steps - processing in the nucleus by the Drosha-DGCR8 complex into pre-miRNAs, followed by export to the cytoplasm and further processing by the Dicer enzyme into mature miRNA. The miRNA is then loaded into the RISC complex containing Argonaute proteins and guides it to target mRNAs to repress translation or promote degradation. MicroRNAs play important roles in various cellular functions and diseases by mediating gene silencing through nine different mechanisms.
Real Time PCR allows for detection and quantification of DNA as amplification occurs. It monitors fluorescence at each cycle to measure DNA accumulation. There are two main types of instrumentation - two-step qRT-PCR which involves reverse transcription followed by PCR, and one-step which combines these steps. Detection relies on fluorescent dyes like SYBR Green or target-specific Taqman probes. Real Time PCR provides advantages over conventional PCR like not requiring gels and being faster and less complex for quantification.
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).
Northern blotting is a technique used to detect specific RNA sequences in a sample. It involves isolating RNA, separating it via gel electrophoresis, transferring it to a membrane, then hybridizing probes with complementary sequences to the target RNA. This allows visualization of gene expression patterns between tissues, developmental stages, and disease states from the detected RNA sequences.
This document provides information about zinc finger proteins. It begins with an introduction to zinc finger motifs, which are protein structural domains characterized by the coordination of zinc ions. The document then discusses the history of zinc finger discovery, functions, and families. It provides details on the most common Cys2His2 zinc finger proteins and their role in DNA recognition and transcriptional regulation. The document also examines uses of zinc finger nucleases for genome editing and their mechanism of action involving creating double-strand breaks in DNA.
Gap-PCR is a PCR technique used to detect deletions or mutations between two known sequences. It uses primer pairs that flank the suspected deletion region to generate products of different sizes depending on whether the deletion is present or not. For example, a normal allele would generate a shorter product than a mutant allele containing the deletion. This allows gap-PCR to efficiently detect deletions larger than 2kb and identify individuals that are heterozygous for the deletion. It has applications in detecting gene deletions associated with conditions like thalassemia.
RNAi – Mechanism and Its Application In Crop Improvementkundan Jadhao
This document summarizes an RNAi presentation on crop improvement using RNA interference. The 3-sentence summary is:
RNA interference (RNAi) is a process of post-transcriptional gene silencing mediated by small RNA molecules. The presentation described the RNAi pathway and various applications of RNAi technology in crop improvement, including increasing nutrient levels, developing virus and pest resistance, and reducing anti-nutritional compounds. Several case studies were provided that demonstrated how RNAi has been used to successfully modify traits in different crops like maize, cotton, coffee, and banana.
Protein-DNA interactions can be either specific or non-specific. Specific interactions involve transcription factors that regulate gene expression by binding to DNA motifs through domains like helix-loop-helix, leucine zipper, or zinc finger motifs. Non-specific interactions involve histones that help structure DNA into nucleosomes within chromatin and can be chemically modified through methylation, demethylation, acetylation, and phosphorylation.
The document summarizes various unsupervised learning algorithms used for analyzing gene expression data from microarray experiments, including k-means clustering, self-organizing maps, and hierarchical clustering. It describes how these algorithms group genes based on similarity in their expression profiles across different conditions or cell types without external labels, helping to simplify data sets and identify genes that may be co-regulated or serve similar functions.
Tamoxifen And CYP2D6: Using Pharmacogenetics to discover a new drugRyan Squire
Dr. Matthew Goetz, assistant professor of oncology and pharmacology at the Mayo Clinic, shared his pharmacogenomic research findings related to risks and occurrence of breast cancer. He explained that in order to truly personalize medicine, you must account for all possible theories and variables. Goetz continued to say that although many believe pharmacology to be boring, it is a key component of the future model of care. Some may say, so this drug doesn’t work–why not just try another drug? It’s much more complicated than that.
Dr. Goetz touched on the variety of cases in his study in breast cancer patients, some with strange and perplexing results. When giving the same drug to multiple patients, each yielded a variety of different results. Some patients had successful reduction in tumor size, while others resulted in no change and some even experienced tumor growth as a result of the drug. Personalized health care is the answer to this, for lack of a better term, ’shot-in-the-dark’ type of therapy. If physicians can understand each patient’s biology and genetic makeup individually, they can better apply treatments and medications. This would therefore reduce health care costs and enable patients to receive much more efficient treatments.
This document discusses pharmacogenomics, which is the study of how an individual's genetic makeup affects their response to drugs. It explains that people can metabolize and respond to drugs differently due to genetic variations. The goal of pharmacogenomics is to optimize drug therapy for each individual by selecting the right drug, dose, and time based on their genotype to maximize effectiveness and minimize side effects. It outlines how variations can influence whether receptors are present to bind drugs, other physiological traits, and how the body processes drugs through absorption, distribution, metabolism and excretion.
Это генетическое исследование, в ходе которого определяется персональный риск возникновения более чем 50 заболеваний, а также чувствительность организма к определенным лекарствам, индивидуальные физические характеристики.
Полученная информация позволит грамотно планировать свой образ жизни и более внимательно отнестись к своим индивидуальным физическим и генетическим особенностям, а значит, даст возможность сохранить здоровье на долгие годы, а также применить полученную информацию при оценке и защите рисков с помощью страхования.
Компания "Независимые Страховые Консультанты" предоставляет 5% скидку на исследование.
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.
Pharmacogenomics examines genetic variations that influence individual drug responses. Single nucleotide polymorphisms (SNPs) can predict good, bad, or no response to a drug. DNA microarrays efficiently identify SNPs to personalize treatment. This allows excluding non-responsive patients from trials, improving drug safety and efficacy. Currently, pharmacogenomics guides cancer treatment and medications metabolized by cytochrome P450 enzymes to prevent overdosing. Benefits include tailored therapy and safer drugs, though complexity challenges wide implementation.
Single nucleotide polymorphisms (sn ps), haplotypes,Karan Veer Singh
This document provides an overview of SNPs (single nucleotide polymorphisms), including their biological background, terminology, detection techniques, and applications. It defines SNPs as single base changes that occur in at least 1% of a population. SNPs can be harmless, harmful, or latent, and are found primarily in noncoding regions, occurring around every 100-300 bases. The document discusses techniques for detecting known and unknown SNPs, including hybridization methods like microarrays and PCR, as well as enzyme-based techniques like nucleotide extension, cleavage, and ligation. It notes applications of SNPs in areas like gene discovery, disease association studies, diagnostics, and predicting treatment response.
Dr. Adithi S Raghavan presented on the topic of pharmacogenetics. The presentation defined pharmacogenetics and outlined several key points:
1. Pharmacogenetics is the study of genetic basis for variation in drug response. Variation can be due to differences in pharmacokinetics, pharmacodynamics, or idiosyncratic reactions.
2. Twin and family studies show drug metabolism and response are highly heritable. Genetic factors account for much of the interindividual variability in drug response.
3. Important examples of single gene disorders influencing drug response include atypical plasma cholinesterase and malignant hyperthermia.
4. Pharmacogenetic testing is becoming more clinically available
This document discusses pharmacogenetics and how genetic differences can influence drug response. It introduces key concepts like the human genome project, pharmacogenomic effects on drug metabolism and transport, and how genetic testing can help determine who will respond to or be toxic to specific drugs. The goal is to develop personalized medicine where a person's genetic makeup is used to optimize drug selection and dosing for safety and effectiveness.
This document provides an overview of pharmacogenetics and discusses:
1. Pharmacogenetics is the study of how genetic factors influence individual responses to drugs. It considers both environmental and genetic factors that impact drug metabolism and effects.
2. Key concepts include how genetic polymorphisms affect drug metabolizing enzymes and transporters, leading to variability in drug efficacy and risk of adverse reactions between individuals.
3. The field has progressed from early discoveries of genetic disorders affecting drug response to now understanding the effects of common gene variants, with the goal of personalized medicine to optimize drug therapy for each patient.
Presentation on Tamoxifen; prevention and treatment for breast cancer. Prepared for Recent Trends in Therapeutics, CLIN 514, at Humber College, November 2011.
Real-time PCR allows for amplification of DNA to be monitored in real-time through the use of fluorescent dyes. It has advantages over traditional PCR such as a wider dynamic range and faster cycling. There are different fluorescent dyes and probe types that can be used for real-time PCR, including SYBR Green and TaqMan probes, which allow for quantification of amplification. Real-time PCR can be used for both absolute and relative quantification of DNA or RNA targets.
Oncodesign aacr 2018 development of a high throughput in vitro screening pl...Florence Fombertasse
Immunological cell death (ICD) is a form of cancer cell death induced by radiotherapy, photodynamic therapy and a few chemotherapeutic agents such as Doxorubicin, Mitoxantrone, and Oxaliplatin. Unlike apoptosis or necrosis, ICD can induce an effective immune response directed against the tumor whereby both dendritic cells and T lymphocytes are mediators of this response. Dying cancer cells recruit and activate immune cells by releasing damage-associated molecular patterns (DAMPS) that help and promote the immune response to antigenic tumor neo-epitopes. Three key DAMPS are associated with the ICD process: calreticulin exposition on the cell surface, ATP secretion and high-mobility group box 1 (HMGB1) release. In order to identify new therapeutic agents that promote ICD in malignant cells, we developed a screening strategy facilitated by an automated in vitro platform with four assays on three different tumor cell lines (human osteosarcoma U-2 OS, human breast MDA-MB-231 and murine liver Hepa 1-6). ICD inducers Doxorubicin and Mitoxantrone used as positive controls increased ATP secretion by 2 to 10-fold at a non-cytotoxic dose after 72 hours incubation on the three cell lines. Both compounds also increased calreticulin exposition by 2 to 4-fold (determined by immunofluorescence using the Operetta High-Content Imaging System) and HMGB1 release by two-fold on the three cell lines. Here we will present recent data from the screening of Oncodesign’s Nanocyclix® library using this platform to identify novel ICD inducers.
MHY2013 is a novel PPAR pan-agonist that was shown to have beneficial effects on metabolic disorders in mouse models of obesity and diabetes. It activated all PPAR subtypes and increased fatty acid oxidation and energy expenditure in liver, adipose tissue, and skeletal muscle by upregulating genes involved in these pathways. MHY2013 improved insulin sensitivity, lowered blood triglycerides and fatty acids, and reduced hepatic steatosis in obese mice without affecting food intake or body weight. The compound's metabolic effects were mediated through increased levels of the hormones FGF21 and adiponectin.
Comprehensive Investigation of the Utilization of SFC/ESI Positive Mode MS fo...Waters Corporation
This document discusses the use of single column convergence chromatography/mass spectrometry (UPC2/MS) for bioanalytical studies. It provides examples of how UPC2/MS can simplify workflows by reducing sample preparation times through direct injection of extracts and improving selectivity over reversed phase chromatography. UPC2/MS also allows for faster separation of challenging compound classes like isomers and lipids compared to traditional techniques like gas chromatography. The document concludes that UPC2/MS provides an orthogonal separation method and combines multiple techniques into one analytical platform for streamlining quantitative bioanalysis in drug discovery and development.
Stratégies orthogonales pour la caractérisation de glycoprotéines thérapeutiq...Quality Assistance s.a.
Stratégies orthogonales pour la caractérisation de glycoprotéines thérapeutiques par LC/MS par Arnaud Delobel, R&D Director
Visit www.quality-assistance.com for more information
The EpiTect Methyl II PCR Array System provides a simple and reliable method to screen DNA methylation levels of multiple genes simultaneously without bisulfite conversion. It uses selective digestion of genomic DNA with methylation-sensitive and methylation-dependent restriction enzymes, followed by quantitative PCR to determine the relative amounts of methylated and unmethylated DNA for each targeted region. As little as 1 microgram of DNA can profile methylation status of up to 94 genes. The system yields comparable data to bisulfite sequencing and beadchip assays but does not require bisulfite conversion. It is a useful tool for high-throughput screening of DNA methylation biomarkers.
The document summarizes qBiomarker Somatic Mutation PCR Arrays, which are PCR-based assays that can rapidly and accurately detect somatic mutations in cancer samples. The arrays can detect mutations present at as little as 0.01% of DNA in a sample. They have been validated to work reliably with different sample types, including archived samples. The arrays cover a wide range of clinically relevant cancer mutations and allow screening of many mutations simultaneously in a single PCR run. The assays and content were selected based on published data on mutation frequencies and functional significance. The arrays provide a simple method for sensitive somatic mutation profiling to aid cancer research.
EUGM 2014 - Ádám Andor Kelemen (Hungarian Academy of Sciences): Physicochemic...ChemAxon
Our proposed goal was the design of a physicochemical property-based scoring method for fragment-based drug discovery. The method was developed for sorting commercially available and virtual libraries, suitable for compilation of an aminerg-GPCR targeted fragment library. In our study we examined the physichochemical characteristics of GPCR-promiscous fragments, and concluded the essential parameters into – so called – Desirability Functions. The validation of the score was carried out using the fragment-screening data of an existing fragment-GPCR library. Subsequent goal is an design and establishment of an aminerg-GPCR targeted fragment-library, using the Fragment-GPCR-Score, followed by in-vitro fragment-screening campaign on several GPCR-targets.
This document summarizes a study that evaluated gene expression profiling of 84 human drug metabolizing enzymes using a pathway-focused PCR array. Researchers treated human hepatocytes with 3 drugs and used the PCR array to measure mRNA expression. They found high intra- and inter-laboratory reproducibility of the PCR array results based on correlation of ΔCT and ΔΔCT values and overlap of differentially expressed genes between experiments. Comparison to TaqMan data also showed high concordance, validating the PCR array as a reliable tool for quantifying drug metabolizing gene expression.
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.
The DTExtm method allows simultaneous analysis of changes in gene expression levels of 145 ADME-associated genes using a microarray. Total RNA is converted to cDNA and labeled before being hybridized to the DTExtm microarray. The method can survey basal gene expression levels and coordinated changes in expression levels caused by drug treatment in various cell lines and tissues. Rifampicin treatment of human hepatocytes showed induction of genes such as CYP3A4, UGT1A1, and ABCB1 in a coordinated manner mediated by PXR activation. Benzoflavone treatment also coordinately induced CYP1A2, UGT1A1, and ABCC2 through AHR activation. DTE
This document compares different methods for calculating amplification efficiencies in real-time PCR. It finds that the Real-Time PCR Miner method provides the most precise efficiency estimates across three real-time PCR instruments by applying whole kinetic curve fitting and iterative nonlinear regression. In contrast, standard curve methods and other amplification plot methods produce instrument-dependent efficiency values. The Real-Time PCR Miner also has the smallest variability between replicate reactions, making it the best tool for accurate efficiency estimation.
Development of a Multi-Variant Frequency Ladder™ for Next Generation Sequenci...Thermo Fisher Scientific
Increasing adoption of NGS has shed light on the need for more
standardized controls to evaluate and optimize system performance.
Samples containing mutations of interest are difficult to source and cell
line pooling experiments to determine limit of detection require significant
investments of time and money. To simultaneously evaluate variant
calling performance in >200 unique amplicons across 50 genes targeted
by NGS tests, AcroMetrix® has developed a proprietary
genomic/synthetic DNA material containing over 550 mutations as a
mixture of SNV’s indels and MNP’s. The limit of detection was then
determined for >400 variants using multiple platforms. Tumor samples
were diluted with matched normal samples to mimic a range of
frequencies. Linearity between the material and diluted tumor tissue
samples were compared. Overall, highly multiplex controls with tunable
frequencies allow for much more extensive, yet streamlined, assay
evaluation and facilitate implementation and impart confidence to NGS
testing.
The document summarizes research evaluating the performance of TaqMan Low Density Arrays for gene expression analysis. It finds that assays on arrays can discriminate 2-fold changes as well as plate assays, and assay results are highly reproducible both within and across arrays. The study also uses a TaqMan Low Density Human Endogenous Control Array to examine housekeeping gene expression across 32 tissues, identifying genes with consistent expression levels suitable for normalization.
Thermo Fisher Scientific developed 200 TaqMan assays to detect mutations in the CFTR gene. They tested the assays on DNA samples containing known CFTR mutations and achieved high call rates and accuracy. The assays can be used to study CFTR mutations according to population frequency and can detect a variety of mutation types including small and large deletions. Example data is shown for assays detecting homopolymer regions and large deletions.
Differential metabolic activity and discovery of therapeutic targets using su...Joaquin Dopazo
The document discusses obtaining estimations of cell metabolic activities from gene expression data using summarized metabolic pathway models. It describes normalizing gene expression data and using an algorithm to estimate metabolic module activities for two conditions, identifying differentially active modules using statistical tests. Metabolic module activities are correlated with protein activation status and metabolite abundance changes, associated with cancer types and treatment responses, and predictive of cell survival. A web server called Metabolizer is introduced that performs various metabolic pathway analyses on gene expression data.
The RT2Profiler PCR Array allows for the simultaneous analysis of 84 genes related to specific pathways using real-time PCR. It provides consistent and reproducible results that correlate well with other gene expression platforms such as microarrays and TaqMan assays. The document demonstrates the array's wide dynamic range, high amplification efficiency, and ability to accurately identify differentially expressed genes between normal and tumor tissue samples.
This study uses real-time PCR arrays to analyze gene expression profiles in liver cells treated with different diabetes drugs to better understand their toxicity mechanisms. The gene expression profile of cells treated with the withdrawn drug Rezulin (Troglitazone) was found to be distinct from the profiles of cells treated with the safer drugs Avandia (Rosiglitazone) and Actos (Pioglitazone). In particular, two genes were found to have much higher expression levels in cells treated with Rezulin compared to the other drugs, indicating these genes may help explain Rezulin's idiosyncratic liver toxicity. The gene expression profiling approach was able to differentiate the effects of different drugs and holds promise for predicting
separated 33 from 26000+ genetic markers in Melanoma(Skin Cancer) cells that are lending themselves to development of classification models with above 90% accuracy and kappa.
Similar to CYP2D6 Allele Specific Copy Number Analysis Using TaqMan® SNP Genotyping Assays And Digital PCR (20)
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
The document summarizes an analytical validation of the Oncomine Comprehensive Assay v3 (OCAv3) targeted next-generation sequencing panel performed in a CLIA-certified laboratory. The validation assessed analytical sensitivity, specificity, accuracy, and precision using formalin-fixed paraffin-embedded tumor samples and cell lines. Results showed the assay met performance thresholds of 90% or higher for detecting single nucleotide variants, insertions/deletions, copy number variants, and gene fusions across a wide range of variants. Over 2,500 clinical samples were subsequently sequenced with the assay maintaining a 95% success rate and average turnaround time of 10 days.
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.
A panel was developed using the OpenArray platform to profile common respiratory tract pathogens via PCR. Assays were designed to target viral and bacterial sequences with high specificity and strain coverage. The panel demonstrated high specificity when tested against genomic standards. Pre-amplification improved sensitivity by enhancing detection of low copy targets. The panel provides a customizable and high-throughput tool for respiratory infection research.
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
This document describes a workflow for generating clonal CRISPR-edited human induced pluripotent stem cell (hiPSC) lines. Key aspects of the workflow include:
1) Developing a hiPSC line that stably expresses Cas9 to facilitate efficient genome editing.
2) Optimizing delivery methods for CRISPR/Cas9 editing tools and improving single cell clone survival and isolation using laminin-521 and StemFlex medium.
3) Applying the workflow to generate hiPSC lines carrying disease-relevant mutations and testing the cell models in assays, finding increased sensitivity to stress in models of Parkinson's disease.
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.
PPT on Direct Seeded Rice presented at the three-day 'Training and Validation Workshop on Modules of Climate Smart Agriculture (CSA) Technologies in South Asia' workshop on April 22, 2024.
Current Ms word generated power point presentation covers major details about the micronuclei test. It's significance and assays to conduct it. It is used to detect the micronuclei formation inside the cells of nearly every multicellular organism. It's formation takes place during chromosomal sepration at metaphase.
Mending Clothing to Support Sustainable Fashion_CIMaR 2024.pdfSelcen Ozturkcan
Ozturkcan, S., Berndt, A., & Angelakis, A. (2024). Mending clothing to support sustainable fashion. Presented at the 31st Annual Conference by the Consortium for International Marketing Research (CIMaR), 10-13 Jun 2024, University of Gävle, Sweden.
Immersive Learning That Works: Research Grounding and Paths ForwardLeonel Morgado
We will metaverse into the essence of immersive learning, into its three dimensions and conceptual models. This approach encompasses elements from teaching methodologies to social involvement, through organizational concerns and technologies. Challenging the perception of learning as knowledge transfer, we introduce a 'Uses, Practices & Strategies' model operationalized by the 'Immersive Learning Brain' and ‘Immersion Cube’ frameworks. This approach offers a comprehensive guide through the intricacies of immersive educational experiences and spotlighting research frontiers, along the immersion dimensions of system, narrative, and agency. Our discourse extends to stakeholders beyond the academic sphere, addressing the interests of technologists, instructional designers, and policymakers. We span various contexts, from formal education to organizational transformation to the new horizon of an AI-pervasive society. This keynote aims to unite the iLRN community in a collaborative journey towards a future where immersive learning research and practice coalesce, paving the way for innovative educational research and practice landscapes.
The debris of the ‘last major merger’ is dynamically youngSérgio Sacani
The Milky Way’s (MW) inner stellar halo contains an [Fe/H]-rich component with highly eccentric orbits, often referred to as the
‘last major merger.’ Hypotheses for the origin of this component include Gaia-Sausage/Enceladus (GSE), where the progenitor
collided with the MW proto-disc 8–11 Gyr ago, and the Virgo Radial Merger (VRM), where the progenitor collided with the
MW disc within the last 3 Gyr. These two scenarios make different predictions about observable structure in local phase space,
because the morphology of debris depends on how long it has had to phase mix. The recently identified phase-space folds in Gaia
DR3 have positive caustic velocities, making them fundamentally different than the phase-mixed chevrons found in simulations
at late times. Roughly 20 per cent of the stars in the prograde local stellar halo are associated with the observed caustics. Based
on a simple phase-mixing model, the observed number of caustics are consistent with a merger that occurred 1–2 Gyr ago.
We also compare the observed phase-space distribution to FIRE-2 Latte simulations of GSE-like mergers, using a quantitative
measurement of phase mixing (2D causticality). The observed local phase-space distribution best matches the simulated data
1–2 Gyr after collision, and certainly not later than 3 Gyr. This is further evidence that the progenitor of the ‘last major merger’
did not collide with the MW proto-disc at early times, as is thought for the GSE, but instead collided with the MW disc within
the last few Gyr, consistent with the body of work surrounding the VRM.
(June 12, 2024) Webinar: Development of PET theranostics targeting the molecu...Scintica Instrumentation
Targeting Hsp90 and its pathogen Orthologs with Tethered Inhibitors as a Diagnostic and Therapeutic Strategy for cancer and infectious diseases with Dr. Timothy Haystead.
EWOCS-I: The catalog of X-ray sources in Westerlund 1 from the Extended Weste...Sérgio Sacani
Context. With a mass exceeding several 104 M⊙ and a rich and dense population of massive stars, supermassive young star clusters
represent the most massive star-forming environment that is dominated by the feedback from massive stars and gravitational interactions
among stars.
Aims. In this paper we present the Extended Westerlund 1 and 2 Open Clusters Survey (EWOCS) project, which aims to investigate
the influence of the starburst environment on the formation of stars and planets, and on the evolution of both low and high mass stars.
The primary targets of this project are Westerlund 1 and 2, the closest supermassive star clusters to the Sun.
Methods. The project is based primarily on recent observations conducted with the Chandra and JWST observatories. Specifically,
the Chandra survey of Westerlund 1 consists of 36 new ACIS-I observations, nearly co-pointed, for a total exposure time of 1 Msec.
Additionally, we included 8 archival Chandra/ACIS-S observations. This paper presents the resulting catalog of X-ray sources within
and around Westerlund 1. Sources were detected by combining various existing methods, and photon extraction and source validation
were carried out using the ACIS-Extract software.
Results. The EWOCS X-ray catalog comprises 5963 validated sources out of the 9420 initially provided to ACIS-Extract, reaching a
photon flux threshold of approximately 2 × 10−8 photons cm−2
s
−1
. The X-ray sources exhibit a highly concentrated spatial distribution,
with 1075 sources located within the central 1 arcmin. We have successfully detected X-ray emissions from 126 out of the 166 known
massive stars of the cluster, and we have collected over 71 000 photons from the magnetar CXO J164710.20-455217.
Describing and Interpreting an Immersive Learning Case with the Immersion Cub...Leonel Morgado
Current descriptions of immersive learning cases are often difficult or impossible to compare. This is due to a myriad of different options on what details to include, which aspects are relevant, and on the descriptive approaches employed. Also, these aspects often combine very specific details with more general guidelines or indicate intents and rationales without clarifying their implementation. In this paper we provide a method to describe immersive learning cases that is structured to enable comparisons, yet flexible enough to allow researchers and practitioners to decide which aspects to include. This method leverages a taxonomy that classifies educational aspects at three levels (uses, practices, and strategies) and then utilizes two frameworks, the Immersive Learning Brain and the Immersion Cube, to enable a structured description and interpretation of immersive learning cases. The method is then demonstrated on a published immersive learning case on training for wind turbine maintenance using virtual reality. Applying the method results in a structured artifact, the Immersive Learning Case Sheet, that tags the case with its proximal uses, practices, and strategies, and refines the free text case description to ensure that matching details are included. This contribution is thus a case description method in support of future comparative research of immersive learning cases. We then discuss how the resulting description and interpretation can be leveraged to change immersion learning cases, by enriching them (considering low-effort changes or additions) or innovating (exploring more challenging avenues of transformation). The method holds significant promise to support better-grounded research in immersive learning.
Authoring a personal GPT for your research and practice: How we created the Q...Leonel Morgado
Thematic analysis in qualitative research is a time-consuming and systematic task, typically done using teams. Team members must ground their activities on common understandings of the major concepts underlying the thematic analysis, and define criteria for its development. However, conceptual misunderstandings, equivocations, and lack of adherence to criteria are challenges to the quality and speed of this process. Given the distributed and uncertain nature of this process, we wondered if the tasks in thematic analysis could be supported by readily available artificial intelligence chatbots. Our early efforts point to potential benefits: not just saving time in the coding process but better adherence to criteria and grounding, by increasing triangulation between humans and artificial intelligence. This tutorial will provide a description and demonstration of the process we followed, as two academic researchers, to develop a custom ChatGPT to assist with qualitative coding in the thematic data analysis process of immersive learning accounts in a survey of the academic literature: QUAL-E Immersive Learning Thematic Analysis Helper. In the hands-on time, participants will try out QUAL-E and develop their ideas for their own qualitative coding ChatGPT. Participants that have the paid ChatGPT Plus subscription can create a draft of their assistants. The organizers will provide course materials and slide deck that participants will be able to utilize to continue development of their custom GPT. The paid subscription to ChatGPT Plus is not required to participate in this workshop, just for trying out personal GPTs during it.
Authoring a personal GPT for your research and practice: How we created the Q...
CYP2D6 Allele Specific Copy Number Analysis Using TaqMan® SNP Genotyping Assays And Digital PCR
1. CYP2D6 Allele Specific Copy Number Analysis Using
TaqMan® SNP Genotyping Assays And Digital PCR
The world leader in serving scien1ce
Toinette Hartshorne, Ph.D.
Sr. Staff Applications Scientist, Genetic Analysis
Genetic, Medical & Applied Sciences
Thermo Fisher Scientific
2. 2
What Is Pharmacogenomics?
• Pharmacogenomics (PGx) is the study of genetic variation that
determines how individuals respond to specific drugs
• It allows physicians to more accurately determine the right drug and
dosage for the patient (avoid adverse drug reactions (ADRs))
• Becoming increasingly important for pain treatment and psychotropic
and cardiac drug dose management
Figure from www.pharmainfo.net/reviews/role-pharmacogenomics-drug-development.
3. 3
PGx Drug Metabolism Enzymes (DMEs)
DMEs catalyze reactions that affect the absorption, distribution,
metabolism, excretion of drugs
Cytochrome P450 system
• Phase I metabolic system of the liver
• Metabolism of >85% of medications
• Genetic variability affects pharmacokinetics
4. Genotyping Highly Polymorphic CYP2D6 Is Challenging
• > 100 characterized CYP2D6 ‘star’ alleles that can contain multiple polymorphisms
• Genotype analysis requires both SNP genoptyping and CNV analysis
4
• > 80 known CYP2D6 polymorphisms within coding and regulatory regions
• includes SNPs, InDels, CNVs, and gene conversion events
Figure adapted from Nature Reviews Drug Discovery (2004)
5. 5
CYP2D6 Star Allele Haplotypes Can Be Duplicated
• Star (*) alleles are gene-level haplotypes that are associated with DME
phenotypes
• For phenotype interpretation purposes, genotyping results are translated
to star allele nomenclature
• The following CYP2D6 star alleles can be duplicated in individuals:
haplotype Major SNPs
Enzyme
Function Activity score
*1 reference Full 1.0
*2 2850C>T; 4180G>C Full 1.0
*2A -1584C>G; 2850C>T; 4180G>C Full 1.0
*4 100C>T; 1846G>A; 4180G>C None 0
*9 2615_2617delAAG Reduced 0.5
*10 100C>T; 4180G>C Reduced 0.5
*17 1023C>T; 2850C>T; 4180G>C Reduced 0.5
*35 -1584C>G; 31G>A; 2850C>T; 4180G>C Full >=1.0
table data from: www.cypalleles.ki.se
6. 6
CYP2D6 Diplotype Determines Drug Metabolism
Likely phenotype
Activity
score
Genotypes
Examples of
diplotypes
Ultrarapid
metabolizer
>2.0 more than two copies of functional alleles
*1/*1x2,
*1/*2x2
Extensive
metabolizer
1.0-2.0
two full or reduced function alleles or
one full function allele plus either one
nonfunctional or one reduced function allele
*1/*1, *1/*2,
*10/*10,
*1/*4, *10/*5
Intermediate
metabolizer
0.5 one reduced and one nonfunctional allele
*4/*10,
*5/*17
Poor metabolizer 0 no functional alleles
*4/*4, *4/*5,
*5/*5, *4/*6
Predicted metabolizer phenotype is used to determine starting drug dosages.
E.g., this chart is relevant to the metabolism of codeine to morphine by CYP2D6:
• Ultrarapid metabolizers avoid codeine use due to potentially toxic morphine levels.
• Poor metabolizers avoid codeine use due to lack of efficacy.
• Extensive & Intermediate metabolizers use age- & weight-specific dosing
• Intermediate metabolizers may not respond as well as extensive metabolizers
table data from: www.pharmgkb.org
7. 7
Phenotypic Outcomes Can Vary Depending On Duplicated
Allele In Heterozygous Individuals
*10 reduced function allele
*1 full function allele
*1 / *10 x 2
Extensive
metabolizer
*1 x 2 / *10
Ultrarapid
metabolizer
*17 reduced function allele
*4 nonfunctional allele
*4 / *17 x 2
Extensive
metabolizer
*4 x 2 / *17
Intermediate
metabolizer
~1-2% duplicated
alleles
heterozygous for
different functional
classes
Metabolizer status will
depend on which allele has
been duplicated
Example 2 Example 1
8. 8
TaqMan® Pharmacogenomics Experiment Workflow
SNP Genotyping Analysis
OpenArray® Plate Genotyper™ Software
Copy Number Analysis
96- or 384-well Plate CopyCaller® Software
Star Allele Results
AlleleTyper™
Software
*NEW* Allele-specific Copy Number Analysis
Identify the duplicated allele to enable more
accurate drug metabolizer status prediction
QuantStudio® 3D
Digital PCR System
TaqMan®
SNP Assays
+
~1-2% of samples
9. 9
How Digital PCR Works
Digital PCR is an analytical technique for quantification of
nucleic acid samples based on PCR amplification of single
template molecules
Negative reactions
Preparation Distribution
Count
Negatives
gDNA or cDNA
TaqMan® Assay
TaqMan® Master Mix
PCR Reaction
Output:
# of molecules/μL
10. CYP2D6 Allele-Specific Copy Number dPCR Workflow
10
Mix Load Amplify Read
QuantStudio® 3D
AnalysisSuite™ Software
SpeI
1. Identify samples with CYP2D6
duplications that are
heterozygous for functionally
different alleles
Purpose: alleles
can be distributed
to separate wells
2. Digest gDNAs to separate
tandem duplicated alleles
3. Run digested material with
appropriate TaqMan®
SNP Assays
4. Analyze data
o Review & confirm cluster plots
o Calculate VIC® and FAM™ dye ratios
using data from AnalysisSuite™
11. TaqMan® Drug Metabolism Genotyping Assays tested
11
VIC®
SNP/DME assay rs number common name
C__32407252_30 rs1080985 CYP2D6*2A g. -1584C>G
C__27102414_10 rs1135840 CYP2D6*2 g.4180G>C
C__27102425_10 rs16947 CYP2D6*2 g.2850C>T
C__27102431_D0 rs3892097 CYP2D6*4 g.1846G>A
C__32407229_60 rs72549350 CYP2D6*9 g.2613 2615delAGA
C__11484460_40 rs1065852 CYP2D6*10 g.100C>T
C___2222771_A0 rs28371706 CYP2D6*17 g.1023C>T
C__27102444_80 rs769258 CYP2D6*35 g.31G>A
13. 13
T
Examples of dPCR Clusters Produced with 2 SNP Assays
Run on a 2 Copy and a 3 Copy Sample
C
T
C
T
A A
G
G
empty empty
empty
empty
T/C
T/C
A/G
A/G
14. *17 1023C>T
1 of 3
1 of 2
0 of 3
2 of 3
1 of 2
3 of 3
1 of 2 1 of 2
NA17116 NA17105 NA17155 NA10859 NA17209 NA17155 NA17116 NA17155
14
Accurate Calling of Copy Number Ratios
120
100
80
60
40
20
0
FAM %
FAM % Average 48.48 66.09 0.49 47.42 53.80 99.46 51.08 33.39
FAM % Expected 50.00 66.66 0.00 50.00 50.00 100.00 50.00 33.33
1 of 2
1 of 3
1 of 2
1 of 3
0 of 3
*4 1846G>A
2 of 3
2 of 3 2 of 3
NA17116 NA17105 NA17209 NA17117 NA17116 NA17209 NA17105 NA17117
70
60
50
40
30
20
10
0
FAM %
FAM % Average 49.21 64.69 64.17 65.29 50.56 33.29 33.28 0.36
FAM % Expected 50.00 66.66 66.66 66.66 50.00 33.33 33.33 0.00
15. 15
Summary
Allele-specific copy number analysis using dPCR &
TaqMan® SNP Genotyping Assays is a simple & effective
method for identifying specific duplicated alleles in
heterozygous samples
This method facilitates accurate CYP2D6 allele genotyping & better
prediction of drug metabolizer phenotype.
Other applications for allele-specific copy number analysis
include:
Genomic targets where CNV of particular alleles is functionally
important
Allele-specific gene expression analysis
Focus is on as-CNV by dPCR, which can be used for copy number analysis of specific polymorphisms in genomic or RNA sequences.
Example shown in this presentation is for CYP2D6
Allele specific copy number analysis by digital PCR method
Pharmacogenomics (PGx)
CYP2D6 SNP and CNV analysis
Digital PCR method
Allele specific copy number analysis of key CYP2D6 variants
Intro on PGx
Foundational field in personalized medicine
Drug development, clinical trials
Individualize drug therapy selection
Predict adverse reactions, dosing, response
Identify increased sensitivity to drug interactions
ADRs - Serious outcomes include death, hospitalization, life-threatening, disability, congenital anomaly, etc.
“Streamline clinical decision making by distinguishing in advance those patients most likely to benefit from a given treatment from those who will incur cost and suffer side effects without gaining benefit”
“Enhance medical product development by improving probability of success”
The promise of pharmacogenomics in identifying and responding to efficacy, toxicity and ADR in commonly administered drugs is a huge opportunity that is only just beginning to have impact in the scientific community
Transition to discussing the enzyme system and genetic variability therein that plays major role in pharmacogenetic testing.
85% of medications metabolized through a CYP450 pathway
Assessing genetic variability in key enzymes is foundation of pgx testing
PharmGKB – lists 52 VIP Pharmgenes involved in Phase I or Phase II
ADME: absorption, distribution, metabolism, excretion (pharmacokinetics)
Drug metabolism is biphasic and consists of stepwise biotransformation and synthesis reactions.
Phase 1 (modification/biotransformation) consists of the oxidation (hydroxylation), hydrolysis, or reduction of a lipid-soluble or nonpolar drug.
Carried out by mixed function oxidases, often in the liver. (CYP P450, Flavins, ADH, oxidases)
Phase 2 (conjugation/synthesis) consists of the conjugation of a drug or its metabolite with an endogenous compound (predominantly glycine, sulfate, or glucuronic acid).
Catalysed by transferases; e.g. glutathioneS-transferases (GSTs).
Phase 3 – further modification and excretion
By a variety of membrane transporters of the multidrug resistance protein (MRP) family.
The result of either phase of metabolism is the production of metabolites that are generally more polar than the parent drug and are more readily excreted in the bile or urine. Both phases of biotransformation are the result of drug interaction with enzymes present in plasma, cytoplasm, mitochondria, and endoplasmic reticulum.
Genotyping the CYP2D6 gene is challenging:
CYP2D6 is located in a gene cluster with 2 highly homologous pseudogenes
the gene is highly polymorphic: both SNP genoptying and copy number variation analysis are required
there is significant variation in allele frequencies among people of different geographical origins.
The entire gene can be deleted or duplicated. Several different CYP2D6 allelic variants have been reported to exist as duplications, with from 2–12 tandem copies.
Gene duplication allele frequencies range from about 1 to about 30%, depending on the population.
Gene conversion events refers to the fact that some CYP2D6 alleles contain some CYP2D7 seudogene sequences
Several commonCYP2D6 alleles are comprised of combinations of 3 or more polymorphisms, many of which are shared by as many as 20 different alleles. Select common polymorphisms are shown in the figure.
It is therefore important to genotype relatively large numbers of polymorphisms across the entire CYP2D6 gene and promoter sequence so that haplotype and genotype can be inferred.
CYP2D6 genotypes can usually be unambiguously assigned on the basis of analysis of approximately 20 polymorphic sites.
PGx Nomenclature: Star alleles are haplotypes (polymorphisms are inherited together) that are associated with enzyme function level.
An activity scoring system is used to catalog the level of enzyme function.
For example: *4 alleles are the most common null allele. They are defined by the 1846G>A splicing defect => no enzyme, no activity.
(*4 alleles can be duplicated or found in tandem with alleles that carry pseudogene sequences)
Need allele-specific copy number solution for the major duplicated CYP2D6 alleles (*1, *2, *4, *9, *10, *17, & *35).
You’ll see why this is important in the next slide.
It is the combination of star allele haplotypes, the diplotype, that predicts the DME phenotype of individual.
This dme information is used to recommend appropriate drug doses.
Example: CYP2D6 converts codeine to morphine, which is the effector drug.
Alternates for ultra & poor include morphine & nonopiod analgesics
When an individual carries a duplication allele and is heterozygous for alleles of different functional classes, it is important to know which allele was duplicated to know the phenotype of the individual.
(as illustrated in the next slide)
Scoring: full activity = 1.0
Reduced activity = 0.5
No activity = 0 – includes *4 splicing defect and *5 gene deletion allele
When an individual carries a duplication allele and is heterozygous for alleles of different functional classes, it is important to know which allele was duplicated to know the phenotype of the individual.
Example 1: if the individual carries 2 full function and 1reduced function allele, they are an ultrarapid metabolizer and should not use drugs metabolized by CYP2D6 that could be toxic to them. If they carry 2 reduced and 1 full funciton, they are an extensive metabolizer, they can use such drugs (age/weight/sex – specific dosing)
Example 2: if the individual carries 2 reduced and 1 no function allele, they are an extensive metabolizer; but if they carry 2 no function alleles and 1 reduced they are an intermediate metabolizer and might not respond as well to CYP2D6-metabolized drugs (track their progress).
Shown is the high throughput PGx experiment workflow:
TaqMan SNP genotyping assay (DME collection) run on OA (or 384-well plates) on QS12k Flex
A VIC or FAM TaqMan probe for each SNP allele
analyze genotypes using TaqMan Genotyper
TaqMan Copy Number assays to DME genes with CNV (CYP2D6)
run in duplex rxns with reference assay on plates on QS12k
analyze with CopyCaller software by ddCt (relative dCt) method.
TaqMan SNP genotype and copy number experiment results can be translated to star allele diplotype results using AlleleTyper translation software.
Matches genetic pattern information with a translation table containing star allele diplotype patterns
Reports out the star allele diplotype call for samples
~1-2% of samples may carry duplicated alleles and be heterozygous for alleles of different functional classes.
The duplicated allele cannot always be discerned by translation analsysis.
Developed a new workflow for allele-specific CN analysis using SNP assays and dPCR
Note: no other PGx platform provides allele-specific CN analysis
How is that done? This is a high level schematic of a digital workflow. What’s nice is that the upfront preparation uses all of the standard techniques that are currently employed today for genomic and RNA extraction- if they work for PCR, they’ll work for digital PCR. What that also means is any samples that you might have in your freezer can be immediately analyzed by digital PCR.
What’s different from running a real-time experiment versus a digital experiment is seen in the second step of this schematic. Rather than running a single reaction and driving a Ct (a measurement of fluorescense that’s created as that molecule amplifies), we instead partition that starting material in 10’s to 100’s or thousands of partitions to get to a digital range. The key to digital PCR is that not every partition (reaction) receives a molecule of interest- defined as setting up replicates at a limiting dilution. In the schematic, this is represented by partitions with DNA and some without.
As seen in the third part of the schematic, any partition that receives a molecule will amplify via PCR to a level that is detectable. The amplified targets are represented in red and are called “positive “reactions. The greys do not exhibit amplification as they are empty and are known as “negative” reactions. And based on formulas that have been developed for analyzing Digital PCR, we count the number of negative reactions, apply the formula, and that will tell you how many molecules you have in your sample.
Input concentration is 1ug input per 50uL digest volume. 1:10 dilution prior to dPCR. Final concentration in dPCR is 0.2ug/uL
Note that *2 SNPs are found in many other alleles
Shown are the known * allele diplotypes for these Coriell repository cell line gDNAs.
The major target SNPs for the indicated * alleles listed were tested by allele-specific CN dPCR using SNP assays
- to determine the allele ratios
- the expected ratios of the FAM and VIC – labelled SNP assay probes are shown
We will show example data for the indicated 2 and 3 –copy samples run with assays to *10 100C>T (reduced allele) and *4 1846G>A (nonfunctional allele).
If anyone asks: NA17209 2 + *36 *1/*4-*36
This sample contains 3 CYP2D6 alleles. 2 are full length and 1 (*36 allele) contains gene conversion to pseudogene CYP2D7 sequences in exon 9. *36 is nonfunctional (and found at highest frequency in Asian populations)
We can distinguish full length from *36 alleles using commercial TaqMan copy number assays specific for CYP2D6 exon 9 and intron 2 or intron 6 sequences.
The exon 9 assay amplifies only full length alleles – not *36 alleles – whereas the intron 2 & 6 assays amplify these as well.
Current: manual count of allele plus wells containing both alleles to get totals for each allele.
Then derive the ratio for FAM to VIC
For 2 copy sample: 1:1
For 3 copy sample: 1T:2C for *10 assays and 2A:1G for *4 assay
In progress: algorithm that counts empties and one allele?? (what to say)
Showing the results of duplicate runs (std dev not shown) for 5 of the assays (2 still being optimized??)
Only the FAM % in each sample is shown: average vs expected
50% for 2C and 33% or 67% for 3C; 0% & 100% also tested to determine background.
Actual and expected ratio %s very close
Very easy to identify the duplicated allele in 3C het samples