Webinars

Upcoming:
Improved RNA-Seq with Degraded Inputs and Tumor Profiling using KAPA RNA HyperPrep Kits

January 25, 2017, 11am EST.
Presented by Dr. Nancy Nabilsi, Roche Sequencing and Life Science

Tumor profiling by high-resolution RNA analysis using next-generation sequencing (RNA-seq) is a rapidly growing application in disease research. Unfortunately, the quality of RNA extracted from biological specimens, particularly FFPE samples, is highly variable, and yields are often low, reducing the ability to generate high-quality sequencing libraries.
Join us on Wednesday, January 25th as Dr. Nancy Nabilsi introduces the new KAPA RNA HyperPrep kits for streamlined library preparation. Nancy will highlight considerations when working with degraded RNA samples, and present comparison data for differential gene expression for tumor profiling.

In this webinar we will:

  • Discuss improvements in the library preparation workflow
  • Provide guidance on working with degraded samples
  • Demonstrate improved detection of differential expression in tumor profiling
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For Research Use Only. Not for use in diagnostic procedures.

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Development of a high-throughput method for the screening of a maize mutant collection

Previously recorded on January 11, 2016 at PAG 2016.
Presented by Dr. Jean-Philippe Pichon of Biogemma.
For Research Use Only. Not for use in diagnostic procedures.

Exploiting genetic variability is at the heart of breeding activities. More than a decade ago, Biogemma has contributed to the creation of a maize mutant collection produced by insertion of Mutator transposon: “the Gene Machine”. After years of recurrent gene-per-gene screenings, we devised a procedure that combines molecular approaches described in the literature and specific in-house optimizations. Hence we improved the preparation of NGS library and the PCRs for the selective enrichment of library fragments encompassing Mutator sequences. Furthermore, we developed custom primers for Illumina sequencing in order to optimize the fraction of informative reads. We also completely automated the analysis with a bioinformatic pipeline that manages every analysis step from raw data to the identification of individual plant carrying the putative germinal insertions. Following the initial method development, a large-scale validation step has been carried out on part of the mutant collection and has proven the reliability of the method. The comprehensive characterization of the “Gene Machine” resource once for all will greatly ease and speed up our reverse genetics studies.

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Dissecting genome dosage, structure and function by whole-genome sequencing

Previously recorded on January 11, 2016 at PAG 2016.
Presented by Dr. Ek Han Tan of the Comai Lab of UC Davis.
For Research Use Only. Not for use in diagnostic procedures.

The structure and dosage of an organism’s chromosomes are precisely maintained and, when altered, can have severe phenotypic consequences. While the study of structural chromosomal changes can provide valuable insight on genome function, use of the required tools and assays in a range of species can be challenging. We recently described a sensitive approach that is able to detect altered dosage by using whole genome sequencing. This technique can be applied to data obtained from a wide array of samples derived from either PCR-free, amplified or exome-capture libraries. For example, we were able to detect haplotype-specific aneuploidy resulting from chromothripsis in plants. Further analysis using the resulting dosage plots provided the precise structural rearrangements induced by chromothripsis. We plan to use this method to characterize a population of individuals that have undergone chromothripsis in order to study the effects of these extreme genomic changes across multiple generations.

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Optimization of crude sample PCR and qPCR using Kapa Biosystems’ evolved enzymes

Previously recorded on January 11, 2016 at PAG 2016.
Presented by Heather Whitehorn of Kapa Biosystems
For Research Use Only. Not for use in diagnostic procedures.

With growing demands for high-throughput gene expression and genotyping analyses, real-time PCR applications must enable greater levels of detection and process efficiency. DNA isolation presents a considerable obstacle when optimizing and streamlining such workflows. Silica membrane-based column purification methods have long been employed but impose additional time, cost, and sample loss. Low-cost, semi-crude extraction methods, such as sodium hydroxide and Proteinase K, reduce workflow time; however, carryover contamination and inhibition may lead to inconsistencies. KAPA Express Extract, a novel thermostable protease and buffer system, offers a solution for crude PCR, allowing for the extraction of PCR-ready DNA in as little as 15 minutes. Wild-type enzymes as such as Taq and Tth are not suitable for high-throughput amplification directly from crude samples. Through directed evolution and optimization of buffer composition, we have developed a novel, highly processive third-generation DNA polymerase, KAPA PROBE FORCE. This enzyme is not only capable of crude sample amplification directly from plant, tissue, and blood samples, but also possesses the 5′-3′ exonuclease activity required for probe-based quantitative PCR. The unique characteristics of this evolved enzyme result in robust amplification across a wide range of sample types and methods of extraction and purification. Here, we provide key parameters of optimization of KAPA PROBE FORCE assays with particular focus on crude sample qPCR. Using experimental data, a troubleshooting workflow is presented for a range of challenging sample types.

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Application of NGS in the diagnostic lab on archival FFPE tissues

Previously recorded on November 4, 2015 at AMP 2015.
Presented by Dr. Brian Walker of the Royal Marsden.
For Research Use Only. Not for use in diagnostic procedures.

Applying next generation sequencing to clinical samples is a goal many diagnostic labs wish to attain. Unfortunately, the majority of samples available to these labs are in the form of formalin-fixed paraffin embedded tissue, which is often archival. The chemical preservation of these tissues for histopathology results in modified and degraded DNA which is difficult to work with in a genetics lab.

The Royal Marden and the ICR have developed a protocol that results in high depth, high quality sequencing of FFPE tissue using the KAPA HyperPlus kit and Nimblegen EZ SeqCap reagents. This is achieved within a turn-around time of three weeks with results discussed in Sequencing Tumour Board meeting where options for patient treatment are discussed. This is currently running on two clinical trials, one for gastro-intestinal cancers and the other for breast cancer.

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Optimized library generation for epigenomic analysis of ultra-low input samples

Part I: Low-input high-throughput DNA methylation analysis

Previously recorded on October 9, 2015 at ASHG 2015.
Presented by Ben Berwick of Emory University.
For Research Use Only. Not for use in diagnostic procedures.

DNA methylation is essential for mammalian development and is often dysregulated in disease. Understanding the dynamics of DNA methylation can thus provide insight into disease etiology and serve as a biomarker. Base-pair resolution analysis of DNA methylation is facilitated by sequencing of bisulfite treated DNA, but this process can be laborious and expensive. Reduced representation bisulfite sequencing (RRBS) uses restriction enzymes to enrich parts of the genome where CpG dinucleotides occur, thus decreasing sequencing costs. To profile rare immune cell subsets, RRBS was further optimized for low input samples using a short-adaptor strategy with the KAPA Hyper Prep Kit and HiFi Uracil+ DNA Polymerase. Titration of the adaptor concentration in DNA limiting conditions (10ng DNA) improved conversion efficiency of the reaction and indicated an optimal adaptor-to-DNA molar ratio of more than >500X. In contrast, no improved reaction efficiency was observed above 50X for non-limiting input concentrations (500ng). Assay specificity and DNA methylation calls were indistinguishable in DNA-limiting conditions as compared to non-limiting conditions. These data provide a robust methodology for RRBS analysis in sample limiting conditions using the Kapa Biosystems efficient chemistry. Resultantly, we analyzed progressive stages in lymphocyte differentiation revealing a dynamic DNA hypomethylation and highlighting the utility of RRBS.

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Optimized library generation for epigenomic analysis of ultra-low input samples

Part II: Low-input ChIP-seq

Previously recorded on October 9th at ASHG 2015.
Presented by Dr. Christopher Scharer of Emory University
For Research Use Only. Not for use in diagnostic procedures.

Chromatin Immunoprecipitation sequencing (ChIP-seq) is routinely used to map covalent histone modifications and transcription binding sites. Typically, 1 ng or more of post-ChIP DNA is required to generate a sequencing library. However, as the analysis of epigenomes transitions from cell culture to primary cells and tissues, the amount of post-ChIP DNA is often in the picogram range. To address this technical limitation, a range of adapter:DNA molar ratios were titrated to optimize library preparation for 1 ng, 100 pg, and 10 pg of ChIP DNA using the KAPA Biosystems Hyper Prep Kit. The well-defined epigenetic modification H3K4me3 was used for the ChIP assay due to its localization to transcription start sites, thus providing a useful QC metric for non-specific enrichment. Optimal adapter concentrations were identified that maximized conversion efficiency and library yield while minimizing adapter dimer carry over. Sequencing of ChIP-seq libraries for each input DNA amount identified a highly significant overlap between libraries prepared from 1 ng, 100 pg and 10 pg of ChIP DNA. The 10 pg libraries were the most variable between technical replicates and identified fewer overall peaks, indicating an increase in the false-negative rate with lower input amounts. These data provide a technical resource for the generation of ChIP-seq libraries from picogram amounts of DNA and highlight limitations to interpretation of these data sets.

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Optimized library generation for epigenomic analysis of ultra-low input samples

Part III: Assay for Transposase Accessible Chromatin sequencing (ATAC-seq)

Previously recorded on October 9th at ASHG 2015.
Presented by Dr. Christopher Scharer of Emory University
For Research Use Only. Not for use in diagnostic procedures.

The accessibility of DNA to proteins such as transcription factors and polymerases has long been a surrogate for regulatory activity. The recent description of the Assay for Transposase Accessible Chromatin sequencing (ATAC-seq) has transformed the analysis of gene regulation by allowing highly efficient generation of sequencing libraries that map regions of DNA accessible to proteins, thus identifying active promoters, enhancers, and other cis-regulatory elements. Unlike other epigenome mapping technologies, ATAC-seq requires 50,000 cells or less and is completed in less than one working day. Using the KAPA Biosystems HiFi Polymerase, an optimized ATAC-seq protocol was developed that efficiently amplifies sequencing libraries from 1,000 – 50,000 cells. These results provide a benchmark for using the ATAC-seq assay on limiting numbers of cells.
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KAPA RNA-Seq with RiboErase: Providing a more comprehensive view of the transcriptome

Previously recorded on December 8, 2015.
Presented by Jennifer Pavlica of Kapa Biosystems.
For Research Use Only. Not for use in diagnostic procedures.

Ribosomal RNA (rRNA) accounts for the vast majority of total RNA, but contributes minimally to understanding gene expression. Therefore, efficient removal of ribosomal RNA (rRNA) is critical in RNA-Seq library preparation to maximize the yield of less abundant, biologically-informative, transcriptome-derived reads. With the KAPA RiboErase Kit, we have developed a flexible and highly effective product for targeted enzymatic depletion of cytoplasmic and mitochondrial rRNA, utilizing complementary oligonucleotide probes and RNase H degradation. In this webinar, we compare this workflow to other RNA enrichment technologies enabling the removal of rRNA, as well as differences in their resulting data quality. Lastly, we will review some important metrics which provide early indications of overall library quality prior to sequencing.

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Mouse genotyping evolved: Overcoming PCR inhibition and accelerate results with crude sample workflows

Previously recorded on September 17, 2015.
Presented by Penny Smorenburg of Kapa Biosystems.
For Research Use Only. Not for use in diagnostic procedures.

Transgenic mice are an essential tool for modeling and evaluating treatments of human diseases. Routine genotyping workflows present a challenge as time and cost must be balanced with assay robustness and reproducibility. Penny Smorenburg discusses novel evolved enzymes and optimized workflows that overcome PCR inhibition across a variety of sample types and targets, enabling streamlined sample-to-genotype PCR and qPCR workflows in less than 1.5 hrs.

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FOG2015

KAPA HyperPlus: The next step in the evolution of library preparation

Previously recorded on June 24, 2015 at the Festival of Genomics.
Presented by Dr. Adriana Geldart of Kapa Biosystems.
For Research Use Only. Not for use in diagnostic procedures.

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NGS library preparation: Theory and practice

Previously recorded on February 17,2015.
Presented by Dr. Maryke Appel of Kapa Biosystems.
For Research Use Only. Not for use in diagnostic procedures.

Part 1: The objectives of library construction


PrepBootcamp-pt1

Part 2: How do I make the best possible library?


PrepBootcamp-pt2

Part 3: A practical example—Library preparation with the KAPA HyperPlus Kit


PrepBootcamp-pt3

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Optimized Library Construction for High-quality Transcriptome Sequencing on the Illumina platform

Optimized library construction for high-quality transcriptome sequencing on the Illumina® platform

Previously recorded on January 22, 2015.
Presented by Dr. Adriana Geldart of Kapa Biosystems.
For Research Use Only. Not for use in diagnostic procedures.

High-quality library preparation is key in transcriptome sequencing to ensure that the data acquired at the end of sequencing is accurate. Dr. Geldart presents data on the ideal quality control metrics to use when preparing RNA-Seq libraries, and show that these metrics assist in predicting the outcome of the sequencing run. We would like to thank Dr. Tom Westerling for his collaboration in this webinar.

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Increasing percentage of analyzed patients using a highly streamlined library prep workflow for exome and long-insert libraries

Previously recorded on December 17, 2014.
Presented by Dr. Jonathan J. Keats of the Translational Genomics Research Institute.
For Research Use Only. Not for use in diagnostic procedures.

Multiple myeloma is a malignancy characterized by the accumulation of plasma cells in the bone marrow. This tumor expansion results in a suppressed white blood cell system, anemia, severe osteoporosis, and compromises kidney function. Recent advances in next generation sequencing can now identify nearly all genetics events existing in an individual case of multiple myeloma.

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FFPE_webinar_Title

FFPE library construction for Illumina® sequencing: Insights and improvements

Previously recorded on May 1, 2014.
Presented by Maryke Appel, PhD of Kapa Biosystems
For Research Use Only. Not for use in diagnostic procedures.

Formalin-fixed paraffin embedded (FFPE) tissue is an important source of DNA for cancer genomics studies and clinical diagnostics. One of the major challenges of high-throughput sequencing is the ability to process low-input samples of variable quality with predictable success rates in standard sample preparation pipelines.

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Meeting the challenges of high-throughput, low-input
library construction for Illumina sequencing

Challenges_1

Presented by Maryke Appel of Kapa Biosystems.
For Research Use Only. Not for use in diagnostic procedures.

Part 1: (18 minutes)

Dr. Appel discusses evolved enzymes and optimized protocols to achieve high library diversity and low-bias library amplification, while meeting challenges such as:

  • Maximizing yield of adapter-ligated libraries
  • Minimizing the number of library amplification cycles
  • Reducing amplification bias, PCR duplication rates, substitutions and other amplification artifacts

Challenges_2Part 2: (16 minutes)

Dr. Appel discusses building a protocol that is scalable over a wide range of DNA inputs and also automation friendly. She introduces case studies of four liquid handling platforms:

  • Caliper Sciclone NGS Workstation
  • Beckman Coulter Biomek FXP
  • Agilent Bravo B NGS Workstation
  • Eppendorf epMotion 5075 TMX

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