NGS Core
(Core-Facility „Omics“)

Prof. Dr. Michael Rehli

 

The „Next Generation Sequencing“ Core of the RCI (RCI NGS Core) provides easy and fast access to the newest sequencing technologies for researchers of the RCI, the University Hospital of Regensburg and the University of Regensburg (in collaboration with the Genomic Core Unit (GCU) University of Regensburg).

We offer a wide range of analyses: gene expression, genome sequencing, immune cell receptor repertoires, chromatin structures and epigenetic modifications. These methods are available for bulk samples but in part also on a single cell basis.

In addition, we also offer support during the planning phase of your NGS experiments (in collaboration with the Computational Core Unit (CCU) of the University of Regensburg) as well as the processing and computational analyses of the generated data.

NEWS

NGS-SERVICE (Illumina NextSeq 550)

General Information

We are an academic sequencing core unit and offer a broad spectrum of NGS-methods. Together with the Computational Core Unit (CCU) of the University of Regensburg we also support you with bioinformatic analyses.

If you are working with us for the first time, please, take the time to discuss your planned experimental setup with us. In case you have questions regarding the process of sample submission and processing, please contact us: genomics-core(at)rcii.de

Prerequisites for using the NGS-Service

  • The sequencing service is limited to research-use only, no warranty or liability apply.
  • The expenses for sample preparation, library preparation and sequencing will be covered by the institution or the principal investigator (PI) requesting the service.
  • Samples and sequencing libraries will only be processed by the RCI NGS Core, if all legal requirements (patient consent complying with the DS-GVO, ethical vote or animal test request) are fulfilled.
  • The submitter will supply a sample sheet for subsequent demultiplexing and trouble shooting.
  • In the case of publication of the sequencing data, the author must acknowledge the service of the RCI NGS Core (i.e.: „Sequencing was conducted at the NGS Core Unit of the Regensburg Center for Interventional Immunology (RCI, University Regensburg and University Medical Center Regensburg, Germany))“

Further terms and conditions apply – please see Terms of Service.

Requirements for Sequencing

NextSeq 550:

  • Flow cells:
    all users must purchase and store the respective number of flow cells for the NextSeq 550 Sequencer (Illumina) on their own. For RCI members the RCI NGS Core will store the flow cells. Important notes on how to order and store a flow cell can be found here[link].

  • Arranging a date for the sequencing run:
    Please, suggest a suitable date for your sequencing run to the NGS Core by consulting the NextSeq 550 Calender [link] for free slots. Then send the proposal to genomics-core(at)rcii.de.

    • Booking Form:
      When the date of your sequencing run is confirmed, complete the booking form and send it in advance to genomics-core(at)rcii.de: BookingNextSeq_Form.pdf (digitally fillable)

    • In addition, you must provide us with a copy of the NextSeq Booking form signed by the responsible PI at the latest on the scheduled date of your sequencing run.
  • Sample Sheet:
    Please, prepare a sample sheet (llumina Experiment Manager) and send it to genomics-core(at)rcii.de

In case you are new to the NGS Core Service, please, inquire which flow cell specifications are required to sequence your experiment on our NextSeq 550. (contact:genomics-core(at)rcii.de)

On the scheduled date of the sequencing run

Please, prepare a Sequencing Library at a concentration of 4 nM (optimal) and take it to the RCI NGS Core. Lower or higher concentrations (0,5 bis 4 nM) are possible, but commonly cause reduced cluster density on the flow cell.

We require a copy of NextSeq Booking form signed by your PI in order to perform the sequencing run!

Data transfer

We will transfer the data in the agreed format after completion of the sequencing run (Options: hard drive or transfer to a sftp server)

In case of questions, please, do not hesitate to contact us at genomics-core(at)rcii.de.

COLLABORATIVE SEQUENCING PROJECTS

General Information

In addition to the plain NGS-SERVICE described above, we also offer – on a collaborative basis – full preparation of the samples starting from tissues or cells.

Depending on your needs and our capacities, we will extract nucleic acids, prepare sequencing libraries, carry out quantification as well as quality control and perform bioinformatic analyses (see general information/methods).

If you have any questions, please do not hesitate to contact us: genomics-core(at)rcii.de

Prerequisites for Collaborative Sequencing Projects

Collaboration partners commit to the following rules:

  • They discuss in advance the experimental setup and the statistical and bioinformatic analyses planned with the RCI NGS Core Facility, to ensure the best possible outcome. This is important as the results from the discussion might influence the sampling strategy, batches for downstream processing and sample preservation options.

  • The head of the RCI NGS Core (Michael Rehli) has to be informed prior to submissions of publications, which derive from collaborative sequencing projects (email: title, list of all authors and abstract). Depending on the contribution of the RCI NGS Core to the project the members of the RCI NGS Core will become coauthors of the publication. In cases where contributions by the RCI NGS Core are considered minor, the author must at least acknowledge the service of the RCI NGS Core (i.e.: „Sequencing was conducted at the NGS Core Unit of the Regensburg Center for Interventional Immunology (RCI, University Regensburg and University Medical Center Regensburg, Germany))“

Further terms and conditions apply – please see Terms of Service.

Sample Submission

Previous to sample submission, please, inform yourself by reading the general information and the notes in the NGS-SERVICE sections. Subsequently, we can arrange a date for the submission of your samples and the signed booking form. Please, contact us ( genomics-core(at)rcii.de). In case you have questions regarding the experimental design, it is your first time collaborating with us or you have a really complex biological question that you want to address with sequencing technologies, please, ask for an extended meeting with our team.


 

DOCUMENTS:

SAMPLE TUBE LABELING:

Please, label the samples in a legible and clear way, with a permanent marker pen. Labels may contain simple letter and number combinations. Please use project ID “PR###-sample number” in collaborative sequencing projects.

SAMPLE MATERIAL:

Next generation sequencing libraries:

  • 4 nM (optimal)
  • 0,5 bis 4 nM (possible)

Cells: in RLT buffer, N2-pellets, crosslinked N2-pellets etc.

  • multiwell-plates or tubes
  • any S1 material will be processed, please inquire for processing of S2 material.

Nucleic acids:

  • DNA: 10 ng - 1000 ng
  • RNA: 10 pg - 1000 ng

Equipment

Next Generation Sequencing

NextSeq 550 (Illumina) High throughput sequencing by synthesis

  • Sequencing 15-26 h
  • 1x75 bp : 10 Mio reads, ~40 Samples 11 h (single-end, high output flowcell)
  • 2x75 bp: 25 Mio reads, ~16 Samples 18 h (paired-end, high output flowcell)
  • 2x150 bp: (paired-end, mid and high output flowcell)
  • High throughput sequencing of large projects will be performed in commercial sequencing centers to ensure cost efficiency. We offer long reads (i.e. 600 PE) by MiSeq (Illumina) sequencing in cooperation with the Department for Biochemistry I of the University of Regensburg.

Single cell analysis

  • Chromium System (10x Genomics) Barcoding of single cells or single nuclei to analyse gene expression, T-cell repertoire or open chromatin.
  • „combinatorial indexing“: (planned)

Quantification and Quality Control

  • Tapestation 2200/4150 (Agilent): automatic gel-electrophoresis in a „microfluidic device“. Quantification and length measurement of nucleic acids.
  • Qubit Fluorometer (ThermoFischer Scientific): Fluorescence-based, exact quantification of RNA, DNA and proteins.
  • KAPA Library Quant Kits (Roche)

Other Analysis Systems and Devices

MassARRAY System

  • SNP analysis by means of iPlex
  • DNA methylation analysis by means of Epityper

 

 

 

 

 


Nucleic acid fragmentation

  • Covaris S220 focused-ultrasonicator
  • Branson SONIFIER 250

Transfection

  • Neon electroporator (low cell numbers)
  • Biorad electroporator (mid to high cell numbers)
  • Amaxa

METHODS

Extraction of nucleic acids

DNA/RNA: nucleic acid adsoprtion based kit systems

Preparation of nucleic acid sequencing libraries (Library Prep)

Sequencing of the immune repertoire of T-cells:

  • T-cell populations
  • single T-cells (10x Genomics)

Sequencing of the transcriptome:

  • strand specific total RNA-seq (Input 100ng -1000ng RNA)
  • strand specific total RNA-seq for low cell numbers (Input 1 to 1000 cells/10 pg to 10 ng RNA)
  • strand specific mRNA-seq (poly-A enrichment) (Input 100ng to 1000ng RNA)
  • strand specific mRNA-seq for low cell numbers (Input 1 to 1000 cells/10 pg bis 10 ng RNA)
  • single cell RNA-seq: 10x Genomics
  • single cell RNA-seq + TCR: 10x Genomics

Sequencing of genomic DNA:

  • whole genome sequencing (WGS)
  • targeted genome sequencing (Exome, custom primer sets)
  • targeted SNP analysis (iPlex) (up to 24 SNPs in 384 sample per run)

Mapping of epigentic characteristics or of chromatin/DNA binding factors:

  • chromatin structure (ATAC)
  • histon modifications or transcription factors (ChIP)
  • DNA methylation (MCIp, bisulfit sequencing)
  • 3D-nuclear structure (HiC, HiChIP)

MassARRAY System

  • DNA methylation analysis by means of Epityper
  • SNP analysis by means of iPlex

PRICES

(in preparation)

Information

RCI NGS Core Team

Prof. Dr. Michael Rehli (head): +49 941 944-38187
Dr. Claudia Gebhard (lab head): +49 941 944-18186
Dr. Nicholas Strieder (bioinformatics): +49 941 944-18188
Johanna Raithel (research associate): +49 941 944-18189

UKR associated team members:
Ute Ackermann
Margit Nützel
Hanna Stanewsky

Publications

Delacher M, Imbusch CD, Hotz-Wagenblatt A, Mallm JP4, Bauer K, Simon M, Riegel D, Rendeiro AF, Bittner S, Sanderink L, Pant A, Schmidleithner L, Braband KL, Echtenachter B, Fischer A, Giunchiglia V, Hoffmann P, Edinger M, Bock C, Rehli M, Brors B, Schmidl C, Feuerer M. (2020). Precursors for Nonlymphoid-Tissue Treg Cells Reside in Secondary Lymphoid Organs and Are Programmed by the Transcription Factor BATF. Immunity S1074-7613(19)30498-4

Minderjahn J, Schmidt A, Fuchs A, Schill R, Raithel J, Babina M, Schmidl C, Gebhard C, Schmidhofer S, Mendes K, Ratermann A, Glatz D, Nuetzel M, Edinger M, Hoffman P, Spang R, Laengst G, Imhof A & Rehli M. (2020). Mechanisms governing the pioneering and redistribution capabilities of the non-classical pioneer PU.1. Nat Commun 11:402

Delacher M, Schmidl C, Herzig Y, Breloer M, Hartmann W, Brunk F, Kägebein D, Träger U, Hofer AC, Bittner S, Weichenhan D, Imbusch CD, Hotz-Wagenblatt A, Hielscher T, Breiling A, Federico G, Gröne HJ, Schmid RM, Rehli M, Abramson J, Feuerer M. (2019). Rbpj expression in regulatory T cells is critical for restraining TH2 responses. Nat Commun 10(1):1621

Kappelmann-Fenzl M, Gebhard C, Matthies AO, Kuphal S, Rehli M, Bosserhoff AK. (2019). C-Jun drives melanoma progression in PTEN wild type melanoma cells. Cell Death Dis. 0(8):584

Gebhard C, Glatz D, Schwarzfischer L, Wimmer J, Stasik S, Nuetzel M, Heudobler D, Andreesen R, Ehninger G, Thiede C, Rehli M. (2019). Profiling of aberrant DNA methylation in acute myeloid leukemia reveals subclasses of CG-rich regions with epigenetic or genetic association. Leukemia 33:26-36

Vatter S, Schmid M, Gebhard C, Mirbeth C, Klobuch S, Rehli M, Herr W, Thomas S. (2018). In-vitro blockade of the CD4 receptor co-signal in antigen-specific T-cell stimulation cultures induces the outgrowth of potent CD4 independent T-cell effectors. J Immunol Methods. 454:80-85

FAQ

How should I acknowledge the RCI NGS Core:

NGS-Service only:

„Sequencing was conducted at the NGS Core Unit of the Regensburg Center for Interventional Immunology (RCI, University Regensburg and University Medical Center Regensburg, Germany)“

Collaborative sequencing projects:

Coauthor list depending on the contributions of different members of the NGS Core, please contact: Michael.Rehli(at)ukr.de

CONTACT

Team:
Prof. Dr. Michael Rehli: +49 941 944-38187
Dr. Claudia Gebhard: +49 941 0944-18186
Dr. Nicholas Strieder: +49 941 944-18188

Address:
RCI NGS Core
Forschungsbau: D5
Department of Internal Medicine III
Franz-Josef-Strauß Allee 11
93053 Regensburg
Universität Hospital of Regensburg
phone: +49 941 944-38187

 

 

 

Terms of Service

I hereby authorize the NGS Core Unit of the Regensburg Centre for Interventional Immunology (RCI NGS Core) to perform certain sequencing services on biological material and/or DNA libraries that my lab submits to the RCI NGS Core. I will make sure that all orders and decisions that members of my lab or other persons that act on my behalf communicate to the RCI NGS Core have been authorized by me, and as the Principal Investigator I agree to carry the final responsibility. I also confirm that my lab and my institution will cover the costs for the sequencing (Quotes are available on request).

I confirm that I have read, understood and accepted the following terms and conditions:

  • Submitters may only submit samples and libraries to the RCI NGS Core for which they have the right to perform comprehensive genomic characterization and to access the resulting data. The submitters must hold appropriate ethical approval, must adhere to all regulations and must have confirmed that no laws or regulations exist that would make it illegal or problematic for the RCI NGS Core to perform sequencing‐based analysis of the submitted material or to make the data available to the submitter. Specifically, any biological samples deriving from human subjects (e.g. patient material) may only be submitted to the RCI NGS Core if written informed consent of the donors has been obtained by the submitter. All samples must be labeled with a pseudonym that does not allow the direct identification of human subjects. With the submission of such samples, the submitters ascertain that written consent has been obtained and that the consent form as well as the requested services have been approved by an ethics vote of the appropriate ethics committee according to the declaration of Helsinki.

  • The RCI NGS Core aims for highest quality but cannot guarantee any minimum read count or quality score because these values depend highly on the submitter‐provided material. Technical failures that are the sole responsibility of the RCI NGS Core (e.g. an accidentally dropped flow cell) are repeated free of charge. Failures that are primarily caused by problems with the submitted materials (e.g. a submitter-prepared library of insufficient quality) are billed to the submitter.

  • Prices for “collaborative sequencing” are subsidized by the Regensburg Centre for Interventional Immunology (RCI). To ensure that these subsidies are used in the most effective way, collaborative sequencing comes with certain responsibilities for the submitter: 

  1. Appropriate measures must be taken to ensure that the study stands reasonable chances of success. Specifically, submitters may be requested by the RCI NGS Core to provide a project abstract and/or a concrete bioinformatic analysis plan that documents the feasibility of the approach.

  2. Submitters must provide a sample annotation sheet with detailed documentation of the protocols used, in order to facilitate sequencing operations and error handling.

  3. The RCI NGS Core coordinator (Michael Rehli) must be informed prior to submission of any publications arising from collaborative sequencing (an informal e‐mail with title, author list and abstract of the manuscript is sufficient), and the RCI NGS Core must be acknowledged through a suitable sentence in the acknowledgment section (e.g. “Sequencing was conducted at the NGS Core Unit of the Regensburg Center for Interventional Immunology (RCI, University Regensburg and University Medical Center Regensburg, Germany)”).

  • The sequencing service is currently provided solely for research purposes and "as is", without any form of guarantee or liability.

  • Samples provided for sequencing in the RCI NGS Core remain the property of the submitter. The RCI NGS Core guaranties keeping of samples for a period of three months. Remaining samples may be returned to the submitter.

  • Sequencing data is made available to the submitter in standardized data formats for a period of three months and is then deleted.

  • Submitters will be held responsible for any damage to RCI NGS Core equipment that results from improper usage or transfer of malware (e.g. viruses).

  • Failure to adhere to the terms of conditions may result in a temporary or permanent ban from using certain services provided by the RCI NGS Core.

Maps