This document summarizes different RNA sequencing library preparation methods and their results. It describes using Nextera, TotalScript RNA-Seq Kit, NEXTflex Directional RNA-Seq Kit, ScriptSeq v2 RNA-Seq, and NEBNext Ultra Directional RNA Library Prep Kit to prepare RNA libraries. It then details the results of quality control checks, sequencing output, alignment rates, and levels of antisense transcription detected for each method, finding that the ScriptSeq v2 RNAseq kit was the most suitable.
How to cluster and sequence an ngs library (james hadfield160416)James Hadfield
A presentation for people intersted in understanding how Illumina adapter ligation, clustering ands SBS sequencing work. Follow core-genomics http://core-genomics.blogspot.co.uk/
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.
How to cluster and sequence an ngs library (james hadfield160416)James Hadfield
A presentation for people intersted in understanding how Illumina adapter ligation, clustering ands SBS sequencing work. Follow core-genomics http://core-genomics.blogspot.co.uk/
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 CRISPR-Cas9 system demonstrates unparalleled genome editing efficiency in a broad range of species and cell types, but it suffers from concerns related to target specificity. Modified guide RNAs and mutant Cas9 proteins have been developed to reduce off-target editing but, in many cases, the alterations also significantly reduce on-target editing performance. In this presentation, Dr Chris Vakulskas discusses a novel, high-fidelity Cas9 protein that reduces off-target gene editing, while maintaining high on-target activity. Dr Vakulskas presents data from the development of the new Alt-R® S.p. HiFi Cas9 Nuclease 3NLS and describes its usefulness in mitigating unwanted off-target gene editing, without the issues associated with transfection of plasmid DNA.
Introduction to real-Time Quantitative PCR (qPCR) - Download the slidesQIAGEN
This slidedeck introduces the concepts of real-time PCR and how to conduct a real-time PCR assay. The topics that are covered include an overview of real-time PCR chemistries, protocols, quantification methods, real-time PCR applications and factors for success.
Real-time quantitative PCR (qPCR) is a preferred platform for high throughput gene expression profiling, where large numbers of samples are characterized for hundreds of expression markers. Technically, the qPCR measurements are performed in the same way as when classical qPCR is used to analyze only a few targets per sample, but the higher throughput introduces additional sources of potential confounding variation that must be controlled for. In this presentation, Dr Kubista describes how high throughput qPCR profiling studies are designed. He covers assay optimization and validation, sample quality testing, and how to merge multi-plate measurements into a common analysis. Dr Kubista also discusses how to cost-effectively measure and compensate for background due to genomic DNA.
Struggling with low editing efficiency or delivery problems in primary or difficult-to-transfect cells? In this presentation, learn about the advantages of using a Cas9:crRNA:tracrRNA ribonucleoprotein (RNP) complex for genome editing. We show the benefits of using RNP complexes, including ease of use, limiting off-target effects, and stability. We also present data showing how genome editing efficiency rates are improved by our Cas9 electroporation enhancer. Furthermore, we provide advice on how to optimize transfection using the Alt-R™ CRISPR-Cas9 System in combination with different electroporation methodologies.
qPCR assays using intercalating dyes, such as SYBR® Green dye, are an economical and effective tool for measuring gene expression. To interpret intercalating dye assays, users need to know how to analyze melt curves, and understand the benefits and limitations of melt curve analysis. In this presentation, Nick Downey, PhD, covers melt curve basics and shares examples of multiple peaks due to suboptimal sample prep, primer dimers, and asymmetric GC content of amplicons. He demonstrates troubleshooting strategies. Experienced and novice users will benefit from an overview of uMeltSM software, developed by the Wittwer lab at the University of Utah, that can predict the melt profile of your assay before you run your experiment.
Cancer therapies that target specific pathways can be more effective than established, nonspecific chemotherapy and radiation treatments, and may prevent side effects on healthy tissues. Such targeted therapies can only be applied after underlying gene mutations have been identified. However, detecting low frequency variants from clinically relevant samples poses significant challenges. Specimens are routinely formalin-fixed and paraffin-embedded (FFPE) for histology, which can decrease the efficiency of NGS library preparation. In this presentation, we discuss approaches for extraction of DNA from FFPE samples, and recommend quality control assays to guide parameter selection for library construction and sequencing depth.
Polymerase Chin Reaction is a technique that takes specific sequences of DNA of small and amplifies it to be used for further testing.
it is also said to be as the Invitro Technique.We have seen an photocopy machine in an office, by which we can copy several pages. So, is the PCR machine in a molecular biology laboratory.
PCR is DNA raplication ina test tube.
Dr Kary Mullis developed PCR.
To amplify lot of double stranded DNA molecules with same size and sequence by enzymatic method and cycling condition.
RNA-Seq Analysis: Everything You Always Wanted to Know...and then somebasepairtech
Computational biologist and Basepair founder, Dr. Amit Sinha (@ausinha) helps viewers navigate the world of RNA-Seq analysis. Topics include: Introduction to RNA-Seq, tools and workflows for analysis, visualization and figures, Q & A. More info at: https://www.basepairtech.com/
Odyssey Of The IWGSC Reference Genome Sequence: 12 Years 1 Month 28 Days 11 ...Fabio Caligaris
Presented at Plant Genomics and Gene Editing Congress: Europe. For more information visit: www.global-engage.com
To meet the challenges of sequencing the large, hexaploid genome, the IWGSC focused initially on developing a solid foundation for sequencing that would accommodate any future advancements in sequencing technologies: i.e., producing physical maps for all 21 individual bread wheat chromosomes.
This Gateway Cloning instruction manual reviews:
Recombination Reactions of the GATEWAY™ Cloning System
The GATEWAY LR Cloning Reaction
The GATEWAY BP Cloning Reaction
Generating Entry Clones
Designing Entry Clones for Protein Expression
Location of Translation Start Sequences
Reading Frame
Examples of Protein Expression Constructs
Destination Vectors
GATEWAY Nomenclature
Gateway® recombination cloning technology circumvents traditional restriction enzyme based cloning limitations, enabling you to access virtually any expression system. Widely adopted in the research community with more than 1,500 references since its launch, Gateway® technology makes collaboration across research disciplines easy and convenient and enables access to a multitude of vectors from these research groups for truly multidisciplinary scientific studies.
http://bit.ly/gateway-cloning-technology-instruction-manual
Struggling with low editing efficiency or delivery problems? IDT has developed a simple and affordable CRISPR-Cas9 solution that outperforms other methods. In this presentation we present the advantages of using a Cas9:tracrRNA:crRNA ribonucleoprotein (RNP) complex in genome editing experiments, and explain why it is the most efficient driver for genome editing compared to alternative methods, such as expression plasmids or the use of sgRNAs. We also review RNP delivery using cationic lipids and electroporation, and provide tips for optimized transfection in your system.
Learn about the power of LNA (Locked Nucleic Acid) technology and QIAGEN's LNA enhanced product portfolio for RNA and DNA research. Download the slide deck!
Next-Generation Sequencing an Intro to Tech and Applications: NGS Tech Overvi...QIAGEN
This slidedeck provides a technical overview of DNA/RNA preprocessing, template preparation, sequencing and data analysis. It covers the applications for NGS technologies, including guidelines for how to select the technology that will best address your biological question.
The CRISPR-Cas9 system demonstrates unparalleled genome editing efficiency in a broad range of species and cell types, but it suffers from concerns related to target specificity. Modified guide RNAs and mutant Cas9 proteins have been developed to reduce off-target editing but, in many cases, the alterations also significantly reduce on-target editing performance. In this presentation, Dr Chris Vakulskas discusses a novel, high-fidelity Cas9 protein that reduces off-target gene editing, while maintaining high on-target activity. Dr Vakulskas presents data from the development of the new Alt-R® S.p. HiFi Cas9 Nuclease 3NLS and describes its usefulness in mitigating unwanted off-target gene editing, without the issues associated with transfection of plasmid DNA.
Introduction to real-Time Quantitative PCR (qPCR) - Download the slidesQIAGEN
This slidedeck introduces the concepts of real-time PCR and how to conduct a real-time PCR assay. The topics that are covered include an overview of real-time PCR chemistries, protocols, quantification methods, real-time PCR applications and factors for success.
Real-time quantitative PCR (qPCR) is a preferred platform for high throughput gene expression profiling, where large numbers of samples are characterized for hundreds of expression markers. Technically, the qPCR measurements are performed in the same way as when classical qPCR is used to analyze only a few targets per sample, but the higher throughput introduces additional sources of potential confounding variation that must be controlled for. In this presentation, Dr Kubista describes how high throughput qPCR profiling studies are designed. He covers assay optimization and validation, sample quality testing, and how to merge multi-plate measurements into a common analysis. Dr Kubista also discusses how to cost-effectively measure and compensate for background due to genomic DNA.
Struggling with low editing efficiency or delivery problems in primary or difficult-to-transfect cells? In this presentation, learn about the advantages of using a Cas9:crRNA:tracrRNA ribonucleoprotein (RNP) complex for genome editing. We show the benefits of using RNP complexes, including ease of use, limiting off-target effects, and stability. We also present data showing how genome editing efficiency rates are improved by our Cas9 electroporation enhancer. Furthermore, we provide advice on how to optimize transfection using the Alt-R™ CRISPR-Cas9 System in combination with different electroporation methodologies.
qPCR assays using intercalating dyes, such as SYBR® Green dye, are an economical and effective tool for measuring gene expression. To interpret intercalating dye assays, users need to know how to analyze melt curves, and understand the benefits and limitations of melt curve analysis. In this presentation, Nick Downey, PhD, covers melt curve basics and shares examples of multiple peaks due to suboptimal sample prep, primer dimers, and asymmetric GC content of amplicons. He demonstrates troubleshooting strategies. Experienced and novice users will benefit from an overview of uMeltSM software, developed by the Wittwer lab at the University of Utah, that can predict the melt profile of your assay before you run your experiment.
Cancer therapies that target specific pathways can be more effective than established, nonspecific chemotherapy and radiation treatments, and may prevent side effects on healthy tissues. Such targeted therapies can only be applied after underlying gene mutations have been identified. However, detecting low frequency variants from clinically relevant samples poses significant challenges. Specimens are routinely formalin-fixed and paraffin-embedded (FFPE) for histology, which can decrease the efficiency of NGS library preparation. In this presentation, we discuss approaches for extraction of DNA from FFPE samples, and recommend quality control assays to guide parameter selection for library construction and sequencing depth.
Polymerase Chin Reaction is a technique that takes specific sequences of DNA of small and amplifies it to be used for further testing.
it is also said to be as the Invitro Technique.We have seen an photocopy machine in an office, by which we can copy several pages. So, is the PCR machine in a molecular biology laboratory.
PCR is DNA raplication ina test tube.
Dr Kary Mullis developed PCR.
To amplify lot of double stranded DNA molecules with same size and sequence by enzymatic method and cycling condition.
RNA-Seq Analysis: Everything You Always Wanted to Know...and then somebasepairtech
Computational biologist and Basepair founder, Dr. Amit Sinha (@ausinha) helps viewers navigate the world of RNA-Seq analysis. Topics include: Introduction to RNA-Seq, tools and workflows for analysis, visualization and figures, Q & A. More info at: https://www.basepairtech.com/
Odyssey Of The IWGSC Reference Genome Sequence: 12 Years 1 Month 28 Days 11 ...Fabio Caligaris
Presented at Plant Genomics and Gene Editing Congress: Europe. For more information visit: www.global-engage.com
To meet the challenges of sequencing the large, hexaploid genome, the IWGSC focused initially on developing a solid foundation for sequencing that would accommodate any future advancements in sequencing technologies: i.e., producing physical maps for all 21 individual bread wheat chromosomes.
This Gateway Cloning instruction manual reviews:
Recombination Reactions of the GATEWAY™ Cloning System
The GATEWAY LR Cloning Reaction
The GATEWAY BP Cloning Reaction
Generating Entry Clones
Designing Entry Clones for Protein Expression
Location of Translation Start Sequences
Reading Frame
Examples of Protein Expression Constructs
Destination Vectors
GATEWAY Nomenclature
Gateway® recombination cloning technology circumvents traditional restriction enzyme based cloning limitations, enabling you to access virtually any expression system. Widely adopted in the research community with more than 1,500 references since its launch, Gateway® technology makes collaboration across research disciplines easy and convenient and enables access to a multitude of vectors from these research groups for truly multidisciplinary scientific studies.
http://bit.ly/gateway-cloning-technology-instruction-manual
Struggling with low editing efficiency or delivery problems? IDT has developed a simple and affordable CRISPR-Cas9 solution that outperforms other methods. In this presentation we present the advantages of using a Cas9:tracrRNA:crRNA ribonucleoprotein (RNP) complex in genome editing experiments, and explain why it is the most efficient driver for genome editing compared to alternative methods, such as expression plasmids or the use of sgRNAs. We also review RNP delivery using cationic lipids and electroporation, and provide tips for optimized transfection in your system.
Learn about the power of LNA (Locked Nucleic Acid) technology and QIAGEN's LNA enhanced product portfolio for RNA and DNA research. Download the slide deck!
Next-Generation Sequencing an Intro to Tech and Applications: NGS Tech Overvi...QIAGEN
This slidedeck provides a technical overview of DNA/RNA preprocessing, template preparation, sequencing and data analysis. It covers the applications for NGS technologies, including guidelines for how to select the technology that will best address your biological question.
Part 2 of RNA-seq for DE analysis: Investigating raw dataJoachim Jacob
Second part of the training session 'RNA-seq for Differential expression' analysis. We explain the characteristics of RNA-seq data that allow us to detect differential expression. Interested in following this session? Please contact http://www.jakonix.be/contact.html
Achieve Complete Coverage of the SARS-CoV-2 GenomeCamille Cappello
Utilize multiple overlapping amplicons in a single tube, using a rapid, 2-hour workflow to prepare ready-to-sequence libraries. The PCR1+PCR2 workflow generates robust libraries even from low input quantities of DNA that may be subsequently quantified and normalized with conventional methods such as Qubit® or Agilent Bioanalyzer, or optionally using the included Swift Normalase reagents.
Provides coverage of >99% of the SARS-CoV-2 genome from limited viral titers
Enabling RNA-Seq With Limited RNA Using Whole Transcriptome AmplificationQIAGEN
RNA-Seq was developed to perform transcriptome profiling and provides a highly precise measurement of expression levels of transcripts and their isoforms. Normally, RNA-Seq analysis requires at least 500 ng –1 μg of total RNA. When working with small biopsies, single cells (such as circulating tumor cells), or other limited material, whole transcriptome amplification (WTA) is normally required. Various WTA methods overcome limited RNA availability and enable transcriptome analysis from limited material or even single cells. In standard PCR-based WTA procedures, however, bias from uneven coverage of cDNA regions with high GC or AT content or amplification errors can lead to the loss of transcripts and wrong variant calling. Here, we compare a standard RNA-Seq library preparation method and the REPLI-g RNA library protocol. The REPLI-g procedure is a PCR-free protocol to efficiently generate RNA-Seq libraries from small amounts of RNA or a single cell in 6.5–7 hours. The REPLI-g protocol uses whole transcriptome amplification based on multiple displacement amplification (MDA), combined with an efficient library adaptor ligation procedure, to prepare RNA-Seq libraries from small RNA amounts. The procedure demonstrates high fidelity, minimal bias and retention of sample‘s transcriptional profile. Compared to standard RNA-Seq library prep, the REPLI-g protocol demonstrates similar reproducibility and sensitivity in transcript detection.
CD Genomics provides a fast, one-stop bacterial RNA sequencing solution from the quality control of sample to comprehensive data analysis. Please contact us for more information and a detailed quote.
It contains information about- DNA Sequencing; History and Era sequencing; Next Generation Sequencing- Introduction, Workflow, Illumina/Solexa sequencing, Roche/454 sequencing, Ion Torrent sequencing, ABI-SOLiD sequencing; Comparison between NGS & Sangers and NGS Platforms; Advantages and Applications of NGS; Future Applications of NGS.
17. 2. Gene expression
TopHat is a fast splice junction mapper for RNA-Seq reads. It
aligns RNA-Seq reads to mammalian-sized genomes using the
ultra high-throughput short read aligner Bowtie, and then
analyzes the mapping results to identify splice junctions
between exons
Tophat2
( bowtie2 )
AlignmentFile.bam
18.
19.
20.
21. Sample Note End 1 Sense
End 1
Antisense
End 2 Sense
End 2
Antisense
% of reads
antisense
% of reads
on sense
FimmX NebNext 148 966 1 925 901 2 373 963 129 259 6 94
FimmX
Nextera
(control
method)
4 513 316 4 813 881 4 516 412 4 751 110 50 50
FimmX NextFlex 61 447 863 828 1 120 296 42 519 5 95
FimmX ScriptSeq_v2 11 491 218 577 776 614 082 10 766 027 5 95
FimmX Totalscript 259 488 7 912 948 3 359 435 117 09 2 98
22. The most suitable method:
ScriptSeq v2 RNAseq kit
Strandedness
Read coverage
High-throughput
