“User-friendly, fast, secure QuantSeq gene expression data analysis“
Dalia Daujotyte, Head of Business Development, Lexogen
Tuesday, 1:30 – 2:00 PM @ Lexogen suite #321
QuantSeq 3‘ mRNA-Seq is a tag-counting method for quantification of gene expression using NGS. It enables most cost-efficient RNA-Seq experiment, and now each QuantSeq kit includes an access code for a complementary DE data analysis integrated on BluebeeTM; genomics platform. This preconfigured data analysis pipeline runs fully automated data processing without bioinformatics involvement. After uploading the samples, the analysis will take less than 10 minutes/sample (batch processing is possible). In this talk we will describe the details of the pipeline.
“Digital deconvolution approaches to infer the cell-type distribution from RNA-seq in Neurodegenerative diseases”
Oscar Harari, Assistant Professor, Washington University School of Medicine, USA
Tuesday, 2:00 – 2:30 PM @ Lexogen suite #321
Genetic factors, implicating both neuronal and glial specific pathways, have been identified associated with Alzheimer’s Disease (AD). We leverage transcriptomic cell-type profiling to infer cell-type distributions of a unique collection of human postmortem brain tissue ascertained to represent autosomal dominant early-onset Alzheimer’s Disease mutations. We employed the Lexogen QuantSeq 3’ mRNA-Seq library prep kit to efficiently quantify their gene expression and discovered co-expression networks that cluster the genes harboring these mutations with additional ones that characterize autosomal dominant late- and early-onset AD.
“Spike-In RNA Variants: Transcript isoforms for the evaluation of RNA-Seq experiments” and “SIRV SUITE: A software platform to compare RNA-Seq quality metrics based on external transcript isoform controls”
Lukas Paul, Senior Manager of Scientific Affairs, Lexogen
Wednesday, 5:00 – 5:30 PM @ Lexogen suite #321
Lexogen, a specialized transcriptomics company, addresses this problem by providing Spike-In RNA Variants (SIRVs) which condense the complexity of transcriptomes in a nutshell. Tiny amounts of SIRVs processed as part of the RNA samples allow for the evaluation and monitoring of RNA sequencing with respect to transcript isoform detection and quantification. However, because standards require standardized data processing Lexogen presents the SIRV SUITE, a new software package for SIRV data evaluation. It is easy to operate and can be integrated in every RNA sequencing pipeline. The SIRV SUITE computes the main quality metrics of gene expression measurements like coefficient of deviation, precision, accuracy, and differential accuracy. The results are presented in overview figures, summarized in quality control forms, and saved in a data base. The linked metadata and “RNA-Seq fingerprint” SIRV quality metrics allow to systematically calculate the concordance of RNA sequencing experiments. Data can be compared to references on the bases of meaningful autonomous control subsets. The SIRV SUITE brings together spike-in derived NGS data, annotations, and data evaluation in an easily navigable way.
“Single molecule sequencing for transcriptome analysis of biological samples using SIRVs control”
Paolo Piazza, Head of Imperial BRC Genomics Facility, Imperial College London, UK
Wednesday, 5:30 – 6:00 PM @ Lexogen suite #321
The generation of multiple protein coding RNA species from a single template via alternative splicing is a highly regulated yet still poorly understood phenomenon at whole transcriptomic level. High Throughput Sequencing has provided new tools for these studies, however short read platforms require sophisticated data analysis to predict splicing variants. Single molecule sequencing technologies (PacBio and Oxford Nanopore/ONT) are proving advantageous to investigate structural arrangements of both DNA and RNA by generating much longer reads. In order to test whether splicing variants can be robustly identified using PacBio, ONT, Illumina or a combination of the above, we used these technologies to sequence human embryonic stem cell transcriptomes spiked with Spike-in RNA Variant Control Mixes (Lexogen), a pool of synthetic RNA molecules with tightly controlled abundance and exon arrangements. Here we report our findings and evidence that Hybrid-Seq strategies (PacBio+Illumina and ONT+Illumina) show superior performance over long-reads-only in most transcriptome analyses.
“Getting the most out of gene expression profiling with QuantSeq: high multiplexing, sensitivity, and reproducibility with lowest costs and efforts”
Lukas Paul, Senior Manager of Scientific Affairs, Lexogen
Thursday, 11: 05 – 11:20 AM @ Great Hall 4-6
With the discontinuation of microarray systems, declining sequencing costs, and convenient NGS data evaluation solutions, attention is now on RNA-Seq as the method-of-choice for gene expression (GEX) profiling. Lexogen’s QuantSeq 3’ mRNA-Seq system offers a comprehensive and highly affordable solution for whole transcriptome GEX analyses and has become a widely approved standard for focused researchers, NGS service providers and – due to its suitability for automation – high-throughput screenings alike. The all-in-one protocol starts with total RNA input as low as 100 pg, generates NGS libraries of sequences close to the 3’ end of polyadenylated RNA and provides single and dual indices with up to 4 x 96 barcodes. Since only short 3’ terminal mRNA sequences are required, the protocol also performs well with degraded or FFPE-derived RNA samples. Superior strandedness (> 99.9%) aids in read mapping and resolution of overlapping genes and antisense RNA, and QuantSeq also delivers precise information on polyadenylation sites allowing the quantification of transcription end-site usage. Since only 1 fragment per transcript is generated, FPKM calculations are unnecessary, and a recommended read depth of 2-5 million short, single-end reads per sample is sufficient for expression profiling of eukaryotic transcriptomes. With a price from USD 19.80 per library prep and sequencing costs from USD 5.00 per sample, easy handling and an express protocol (4.5 hours) QuantSeq enables exceptionally precise and cost-effective gene expression experiments. Furthermore, Lexogen’s collaboration with Bluebee now provides QuantSeq users also with a cloud-based, non-expert access to data analysis up to differential gene expression.
“Identification of internal priming events in Lexogen’s QuantSeq library preparation leads to accurate gene quantification and detection of 3’ ends“
Patrick Schagerl, Michael Ante, Dalia Daujotyte, Andreas Tuerk
Tuesday, 1:30 – 2:30 PM @ Great Hall 3
Accurate gene quantification and identification of 3’UTRs is important in many areas of biological research. Lexogen’s QuantSeq library preparation provides an easy and fast protocol for generating highly strand-specific NGS libraries close to the 3’ end of polyadenylated RNAs. The use of oligo(dT) primers in QuantSeq, however, also generates reads at long internal poly(A) stretches. Such reads can negatively affect the accuracy of end site detection and gene quantification. We, therefore, developed an internal priming filter (IPF) to remove reads associated with internal priming events. For this purpose, we investigate properties of the genome sequence and annotation around the read ends. In a window immediately downstream of the read end we count the number of A’s, upstream we search for 3’ motifs. We find the optimal cut-off for the number of A’s by fitting a logistic regression model to sets of high-confidence internal priming and end sites having the same size ratio as internal priming and end sites in one chromosome. This ratio is determined by fitting a Gaussian mixture to the bimodal distribution of A-count frequencies. On the MAQC dataset the IPF retained 85.32% of the original reads in 3’UTRs, whereas only 5% and 3.68% were retained in 5’UTRs and introns, respectively. An increased concentration of reads at the gene ends can also be observed in coverage plots. Base content around priming sites after IPF showed the typical end site signature with an increased frequency of CA at the read end site, A upstream and GU/U downstream. Correlation between log fold changes of read counts and qPCR values increased after IPF from R2=0.7234 to R2=0.8175 suggesting superior accuracy in detecting differentially expressed genes. The described method is part of the QuantSeq pipeline including gene quantification and differential expression analysis which can be downloaded from the Lexogen website.
Monday, 13 February, 2017
Welcome coffee, cake, and ice cream @ 3:00 PM
Tuesday, 14 February, 2017
Paella and Sangria dinner @ 5:30 PM
Austrian Schnapps tasting @ 9:30 PM
Wednesday, 15 February, 2017
Lexogen Blueberry Mojito happy hour
@ 9:30 PM
Win a prize
Solve the Lexogen Quiz, win a prize, and get your lucky lottery ticket!*
Upon successful completion of the quiz, you can get an RNA T-Shirt or Lexogen beach tennis set and the lottery ticket at our suite. You can participate in the quiz until 12 PM on Thursday (February 16).
The lottery ticket will give you a chance to win one of the special prizes. The prize drawing will take place at the Lexogen suite on Thursday (February 16) at 1 PM.
You can access the quiz through the link su.vc/lexogen-quiz.
*Special terms and conditions apply, which are available here.
Meet our team at AGBT!
Stop by our suite or contact us via email for any questions. Our AGBT team is looking forward to meeting you!
CEO and Founder
Head of Business Development
Senior Manager of Scientific Affairs
Business Development Manager UK
North American Sales Manager
Schedule a meeting with us
You are very welcome at our Lexogen suite anytime. If you want to have one-to-one meeting to discuss some particular topics, please send us a request or write to us at firstname.lastname@example.org, specifying your interests. We will make sure that the most experience in this topic team member is available for you.