BioMediTech Research Infrastructure

NGS & Sanger Sequencing

The facility offers the use of Illumina MiSeq sequencer for a wide range of next-generation sequencing. In addition, the facility offers Sanger sequencing for researchers’ ready-made sequencing reactions.

Introduction

Sanger sequencing

Sequencing facility at Kauppi campus is situated in Arvo building 2nd floor, room E215, and it houses Applied Biosystems 3500xL Genetic Analyzer run by a core technician. ABI 3500xL is a 24-capillary instrument capable for running 2 x 96 samples (sequencing or fragment analysis) at a time.

The instrument is operated by the sequencing core staff who will run your ready-made reactions. Our service routinely delivers the results in 24 hours of samples left from Monday to Thursday, provided that the samples with sample list are delivered to sequencing service by 14:30. Results of samples left on Friday afternoon by 14:30 will be ready next Monday morning. In case of any delays we will contact you by email.

At the first time our customer (research group) gets a password protected domain where the results are saved for one month. Be sure to collect your data during that time.

The service is run-only with auto-analysis of raw data from ready-made samples. We recommend usage of ABI BigDye™ v3.1 Chemistry (Sanger sequencing) starting with the manufacturers protocol. Please note that only 0.2 ml MicroAmp™ 8-Tube Strips and 0.2 ml MicroAmp™ Optical 96-Well Reaction Plates can be used in ABI 3500xL.

NGS

Illumina MiSeq sequencer for targeted and small genome sequencing is located in Arvo building 2ndfloor, room E215. The system enables a broad range of applications, from targeted gene sequencing to metagenomics, small genome sequencing, targeted gene expression analysis, and more. Customers prepare the sample libraries and order all the reagents for the instrument by themselves. The instrument is run by facility technician, or by customer after user training provided by the core personnel. The data can be transferred from the MiSeq by BaseSpace or picked directly from the instrument.

Booking system for the instrument: https://next.cirklo.org/biomeditech / (MiSeq). All information about the run is also written down to the logfile next to the instrument.

Read more about the instrument from the manufacturers web site:
http://www.illumina.com/systems/miseq.html

For more information about the applications and prices, please contact Illumina’s Finnish Territory Account Manager Reija Laitinen (Cell: +358 40 521 2847, E-mail: rlaitinen@illumina.com).

ACKNOWLEDGEMENT

All the users of the NGS & Sanger Sequencing facility services are obligated to acknowledge the facility in publications:

“The authors acknowledge the Tampere facility of NGS & Sanger Sequencing for their service.”

Instructions

Sanger sequencing

The customers make the sequencing samples ready for run themselves (cycling, cleaning, and suspension in HiDi Formamide). You can use our convenient Freezer Program to purchase BigDye™ Terminator v3.1 and HiDi.

Fill the sign-up list on the freezer and the products will be charged with your sequencing bill. The Buffer for BDT is available in cold room E218 in aliquots.

Sequencer plates or strips with purified sample in Hi-Di formamide are taken to the cold room (instructions below). Sample sheets are modified by the researcher and then sent in Excel 97-2003 Workbook form (.xls) to sequencing(at)uta.fi. Instructions and links to sheets below.

  • Deliver samples either on 200 µl strips in sets of 24 (n x 24, empty tubes filled with HiDi at least 12 µl /well) or on full 200 µl sequencing plates (96 samples) to a rack inside a blue box in cold room E218.
    • Only MicroAmp™ plastics are allowed for sequencing and fragment analysis. Please note that any other strips or plates cannot be used with ABI 3500xL! Accepted plastics can be purchased from the basic equipment cabinets on 2nd and 3rd floor corridors near the sequencing facilities.
    • Accepted strips are MicroAmp™ 8-Tube Strips, 0.2 ml
    • Accepted plate is MicroAmp™ Optical 96-Well Reaction Plate, 0.2 ml
    • You may use for example MicroAmp™ 8-Cap Strips to seal your strips or plates.
  • Fill in information about your samples to form found on a table outside the cold room. Please, remember also to add your phone number.
  • Each row of the form has a running number. Write this number clearly on a post-it slip found on the shelf and attach the slip to your sample rack.
  • If you are using strips mark the beginning sample number on one end of the strip (1, 9, 17, 25 etc.). Place your strips on the Sequencing service rack starting from A1. (Your 1st sample to position A1, your 9th sample to position A2, etc.)
  • If you are using plate you will be charged for the whole plate (4 x 24 samples). All wells have to be filled with HiDi (at least 12 µl /well). Make sure your sample number 1 is in well A1 on your plate, sample number 2 is in well B1 etc. Place the plate on the Sequencing service rack also starting from A1.
  • Deliver your samples and the sample list by 14:30. Samples are ran in first-come-first-serve basis. In case of backlog the samples and sample list arrived last will be sequenced on following day.

We will send an e-mail when the results are in your own password-protected domain.

Modifying sample sheet

  1. Choose the right sample sheet (Excel):
  1. On Excel, fill the Plate Name on cell A4 in the following way: DDMMYYYY_initials. You may also add an identifier (for example 03092018_NN_exon1). Maximum number of characters is 30.
  2. Add Sample Names from cell B7 downwards and add the Results Group from the cell E7 downwards. (Please note that the name of the Results group is case sensitive.) Add the Owner Name (for example your name).
  • Disallowed characters are: space / ( ) * ” – + : . ? ’ ,| ä ö å
  • In case your group doesn’t exist in Results group list please contact sequencing service.
  1. Unless you have a full plate remove unnecessary information from columns C, D, and F. For example, if you’re running 24 samples, remove data from row 31 downwards by painting the cells C31-F102 and pressing delete button. Leave the column A (Well Positions) untouched.
  2. When the sample sheet is ready, rename it with your Plate Name (DDMMYYY_initials) and send it in table form (.xls) to sequencing(at)uta.fi.

Additional information

Short and long sequences as well as fragment analysis can be run in a same plate as long as there is 24 (x n) of each. Please ask additional information from sequencing service if you need to combine these different sample types in a same run.

The sequencing of long (>850 bp) products will take approximately 2 hours per injection, i.e. 1 x 24 samples. The fragment analysis and the sequencing of shorter products will take approximately 1 hour per one injection (1 x 24 samples).

With ABI 3500xL only MicroAmp™ plastics are accepted. They can be purchased with reduced price from the basic equipment cabinets on 2nd and 3rd floor corridors near the sequencing facilities. Links to manufacturer’s sites:

Prices

Sanger sequencing

Price per each Sequencing run derives from actual operating costs (chemicals, consumables, instrument service contract and staff salary) divided by number of runs in the facility. Please inquire!

NGS

The kits and reagents for MiSeq are provided by users themselves. The costs for Illumina Product Care Service contract will be divided among the users according to the use.

Contacts

Sanger Sequencing Service email:

sequencing(at)uta.fi

Sanger Sequencing Service Manager:

Laboratory Technician
Kati Rouhento
kati.rouhento(at)uta.fi
Tel: +358 50 318 6866

Sanger Sequencing Service Vice Managers:

Hanna Selin
hanna.selin(at)uta.fi
Tel: +358 50 318 2119

Marjo Salminen
marjo.t.salminen(at)uta.fi
Tel: +358 40 190 1507

NGS Service Manager:

Laboratory Technician
Kati Rouhento
kati.rouhento(at)uta.fi
Tel: +358 50 318 6866

Intranet

Intranet (password required)

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