PacBio Iso-Seq

The PacBio Sequel II provides long read capability for sequencing large amplicons, whole transcripts, targeted regions of the genome, and whole genomes. It can also provide base modification signatures for epigenetic studies. The facility had a Sequel from April 2017 to January 2020. Then they acquired the Sequel II. The data is proving to be indispensable for accurate phasing of mutations spaced more than 1kb apart and providing genome structure on a larger scale than is currently possible with short read technologies. The Sequel II SMRT cells have 8 million ZMWs! This is 8X the data from the Sequel I.

GETTING STARTED

General RNA quality guidelines for Iso-Seq preps are as follows:

  • RNA samples should be DNAse treated
    • QC RNA, RIN >7 (ideally >8)
    • A260/A280≥ 1.9
    • Recommends 300ng total RNA input

The input volume for the assay is 7 µL, so for a 300 ng input, the preferred concentration range is approximately 43 – 100 ng/µL. Please err on the side of too concentrated where possible. Keep in mind that we will use approximately 3 µL of the sample for initial RNA QC.

To discuss a project or obtain a quote please e-mail gecinfo@biotech.wisc.edu.

Please review our current sample drop-off guidelines (effective June 2021): GEC Sample Exchange Policy

SAMPLE SUBMISSION

Samples may be submitted on Tuesday and Thursday between 9-10AM by appointment.

Appointments may be made by e-mailing gecinfo@biotech.wisc.edu.

All sample submissions must include completed electronic copies of both the PacBio Iso-Seq Submission Form and the Sample Submission Spreadsheet. If you have any questions please let us know.

DOCUMENTS FOR DOWNLOAD

PacBio Iso-Seq Express library prep Submission Form

GEC Sample Submission Spreadsheet 

Turnaround time for PacBio sequencing is typically 2-3 weeks.

BIOINFORMATICS

Support is available for projects that are run at UWBC facilities, and is provided for a fee by the UWBC Bioinformatics Resource Center. Terminals and software are available in our computer lab for clients of UWBC facilities who are internal to UW-Madison or the University of Wisconsin System who need to perform further analysis but lack the necessary hardware and software.

ACKNOWLEDGING UWBC AND THE GENE EXPRESSION CENTER

A visible measure of a core’s impact is made through proper acknowledgement of usage in publications. To acknowledge our core, please use the following statement: “The author(s) thank the University of Wisconsin-Madison Biotechnology Center Gene Expression Center for providing Iso-Seq library preparation and the DNA Sequencing Facility sequencing services”.

Please cite our Research Resource ID: RRID:SCR_017757

The Sequel II System generates long reads that are well-suited for characterizing full-length transcripts produced from high-quality RNA samples. The Iso-Seq Express Library preparation allows detection of full-length transcripts up to 10kb and supports multiplexing up to 12 samples.
Iso-Seq Express Library Preparation UW System Rate Non UW System Rate
Iso-Seq Sample 1 $1,620 $2,240
Each additional sample $170 $175

Interested in ScISOr-Seq?  E-mail us at gecinfo@biotech.wisc.edu

Pricing effective April 1, 2021.

All data and services provided by the University of Wisconsin Biotechnology Center Gene Expression Center are intended for research purposes only. Services are not intended nor certified for diagnostic or clinical use.