Next Generation Sequencing

  • Genomics’ most flexible and powerful tool.
  • Genomics’ most flexible and powerful tool.
  • Genomics’ most flexible and powerful tool.

The goal of the Next Generation Sequencing (NGS) Core Facility is to provide cutting edge next generation sequencing technology to its users. Next Generation Sequencing has become a key analysis method for biological research. The capacity to expand analysis from more or less defined genomic regions to genome wide studies has boosted the pace of research discovery and enabled researchers to obtain a global view on biological processes. Advice and guidance of sequencing projects are offered by our team that relies on years of experience with sequencing systems and sequencing data analysis. All common sequencing applications are supported and the development of novel methods and protocols encouraged.

Meet Us

Meet us at the Illumina User Group Meeting, 10-11 March 2015, Heidelberg, Germany

Services

    Library Preparation

    Library Preparation

    A prerequisite for NGS is the generation of so-called sequencing libraries out of the correspondent DNA or RNA sample. It consists mainly in the ligation of adaptors, which make the sequencing reaction possible. There are many protocols and kits available depending on sample source and biological question of interest.

    The Next Generation Sequencing Core Facility offers two major pipelines:

    • DNA preparation - suitable for all types of DNA samples e.g. ChIP, gDNA, cDNA, amplicons
    • RNA preparation - applications: sequencing of total RNA, purified mRNA or ribosomal-depleted RNA

    Preparation of libraries by user is encouraged and supported.

    Quality Control

    Quality Control

    Every sequencing library, prepared by facility or user has to be carefully quality checked. This includes three major steps:

    • Measurement of concentration (Fluorescence NanoDrop)
    • Measurement of size (Bioanalyzer, Fragment Analyzer)
    • Mimicking cluster formation (RT-PCR)

    Sequencing

    Sequencing

    Sequencing is performed on Illumina HiSeq 2500 instruments with the capacity to produce up to 70Gbp per lane. The request unit is one sequencing lane, which can be filled up (multiplexed) with as many samples as the user requires. Sequencing read length (50-500bp) and modus (single read or paired end) can be freely chosen depending on application.

    Currently we offer the following read length modes, the preferred mode is given in brackets:

    • Single Read 50 bp (V4)
    • Paired End 50 bp (Rapid Mode)
    • Single Read 100 bp (Rapid Mode)
    • Paired End 125 bp (V4)
    • Paired End 150 bp (Rapid Mode)

    Data Analysis

    Data Analysis

    Primary data output from the Illumina sequencers is converted to compressed BAM files that hold sequences from both reads (in case of paired end sequencing), quality information, index read barcodes and other information. We generate quality metrics from the sequencing process and analyse the reads using Fastqc (Basecalling quality, GC content, overrepresented sequences, …) and PreSeq (duplications and library complexity) to check successful sequencing and potential downstream analysis problems. Lanes running pools of multiple samples are demultiplexed by the index barcode into one file per sample and their quality is checked individually.

    On demand we offer mapping to reference genomes of model organisms either with Bowtie (ChIP-Seq), Tophat/STAR (RNA-Seq) or BWA (Whole Genome, genotyping and SNP calling). The alignment data can be visualized in a genome browser (IGB, IGV, UCSC) and further analyzed in Galaxy.

    Resources

    Sequencers

    Currently in operation:

    Lab equipment

    Software and Data management

    • Forskalle - For submission and administration of your sequencing requests.
      http://ngs.csf.ac.at/forskalle
    • Queue - A page to check the progress of your requests and securely download finished sequencing data. http://ngs.csf.ac.at/
    • Galaxy - Galaxy allows our customers to perform basic bioinfomatics tasks. In collaboration with the GenAU bionformatics group we provide some custom tailored tools for groups adressing their research.
      http://ngs.csf.ac.at/galaxy
    • Reserva - Book a time slot for the Covaris DNA shearer.
      http://ngs.csf.ac.at/reserva
    • BAM2FQ - A light-weight tool to convert unaligned BAM files to fastq format. Just drag and drop your BAM files into the window. It detects paired end runs and splits them automatically into two files. Runs on any operating system with Java installed.
      http://ngs.csf.ac.at/tools/bam2fq.jar
    • CSF-NGS Github - Illumina-related tools and snippets for quality metrics and file conversion.
      https://github.com/csf-ngs

    Protocols

    User Information

    Access

    The NGS Core Facility offers access to its services to all members of the Campus Vienna Biocenter. Unused capacity is offered to external Users. In order to estimate feasibility and allocable resources we strongly recommend to contact our team when planning a sequencing experiment.Queuing follows first come, first serves policy.

    All relevant information for Users can be found in our User Guide which can be downloaded from Forskalle. (http://ngs.csf.ac.at/forskalle/)

    Additional information is in the slides from the Introduction to Forskalle presentation.

     

     

    Citing the facility

    How should I acknowledge The CSF NGSequencing Service?

    We all like to receive credit for the work we do don't we?

    Continuation of funding for the CSF NGS facility by the city of Vienna and the ministry of science depends on documented evidence of contribution to scientific output.

    We therefore demand acknowledgement of utilization of the facility in publishing work.

    A simple statement is sufficient and can either be placed in the materials and methods section or at the end of your manuscript (acknowledgements) depending on journal format.

    Suggested minimal format:

    Solexa (Deep, Next generation, or however you named it in your manuscript) Sequencing was performed at the CSF NGS Unit (csf.ac.at).

    Your sample will be then listed on our website:

    www.csf.ac.at/facilities/ngs/scientific-contributions/

    Terms & Conditions

    All requests submitted to the CSF NGS are subject to the general cooperation conditions of the CSF as well as CSF NGS policy if no additional agreement exists.

    • User Guide (link to pdf coming soon)
    • Price List (link to pdf coming soon)
    • general Cooperation Conditions (link to pdf coming soon)

    Publications

    A DEK Domain-Containing Protein Modulates Chromatin Structure and Function in Arabidopsis.
    Waidmann S, Kusenda B, Mayerhofer J, Mechtler K, Jonak C.
    Plant Cell. 2014 Nov;26(11):4328-44. (abstract)

    Role of STN1 and DNA Polymerase α in Telomere Stability and Genome-Wide Replication in Arabidopsis.
    Derboven E, Ekker H, Kusenda B, Bulankova P, Riha K.
    PLoS Genet. 2014 Oct. 9. (abstract)

    SNW1 enables sister chromatid cohesion by mediating the splicing of sororin and APC2 pre-mRNAs.
    van der Lelij P, Stocsits RR, Ladurner R, Petzold G, Kreidl E, Koch B, Schmitz J, Neumann B, Ellenberg J, Peters JM.
    EMBO J. 2014 Nov 18. (abstract)

    ADAR2 induces reproducible changes in sequence and abundance of mature microRNAs in the mouse brain.
    Vesely C, Tauber S, Sedlazeck FJ, Tajaddod M, von Haeseler A, Jantsch MF.
    Nucleic Acids Res. 2014 Sept. (abstract)

    Cis-Regulatory Requirements for Tissue-Specific Programs of the Circadian Clock.
    Meireles-Filho AC, Bardet AF, Yáñez-Cuna JO, Stampfel G, Stark A.
    Curr Biol. 2013 Dez. (Epub) (abstract)

    Transcriptome and proteome quantification of a tumor model provides novel insights into post-transcriptional gene regulation.
    Jüschke C, Dohnal I, Pichler P, Harzer H, Swart R, Ammerer G, Mechtler K, Knoblich JA.
    Genome Biol. 2013 Nov. (abstract)

    A Histone Deacetylase Adjusts Transcription Kinetics at Coding Sequences during Candida albicans Morphogenesis.
    Hnisz D, Bardet AF, Nobile CJ, Petryshyn A, Glaser W, Schöck U, Stark A, Kuchler K.
    PLoS Genetics 2012 Dec. (abstract)

    An Unusual Case of Apparent Hypermutation in Arabidopsis thaliana.
    Sasaki T, Naumann U, Forai P, Matzke AJ, Matzke M.
    Genetics. 2012 Dec. (abstract)

    Forward and Reverse Genetics through Derivation of Haploid Mouse Embryonic Stem Cells.
    Elling U, Taubenschmid J, Wirnsberger G, O'Malley R, Demers SP, Vanhaelen Q, Shukalyuk AI, Schmauss G, Schramek D, Schnuetgen F, von Melchner H, Ecker JR, Stanford WL, Zuber J, Stark A, Penninger JM.
    Cell Stem Cell 2011 Dec. (abstract)

    A computational pipeline for comparative ChIP-seq analyses.
    Bardet AF, He Q, Zeitlinger J, Stark A.
    Nat. Protoc. 2011 Dec. (abstract)

    Team

    Andreas Sommer

    Andreas Sommer

    Core Facility Head
    VBC 2 / PG 4

    Laura-Maria Bayer

    Laura-Maria Bayer

    Sequencing Specialist
    IMBA-GMI / 5.55

    Carmen Czepe

    Carmen Czepe

    Sequencing Specialist
    IMBA-GMI / 5.55

    Heinz Ekker

    Heinz Ekker

    Bioinformatician
    VBC 2/PG 4

    Bartlomiej Gebarski

    Bartlomiej Gebarski

    Sequencing Specialist
    IMBA-GMI / 4.72.2

    Renate Landwehr

    Renate Landwehr

    Sequencing Specialist
    IMBA-GMI / 4.72.2

    Ido Tamir

    Ido Tamir

    Bioinformatician
    VBC 2/PG 4

    Contact