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.

We provide PacBio whole transcriptome and  single cell transcriptome library preparation services.  For whole transcriptome we require a minimum of 400ng total RNA and for single cell transcriptome we require 75ng 10X Genomics 3′ scRNA cDNA template.

For new clients we recommend a meeting is  scheduled with us to discuss your project before submission.  Please email gecinfo@biotech.wisc.edu.

The following are our recommendations for RNA quality metrics:

1) Strongly recommend RNA samples are DNase treated
2) A260/A280 ratio range 1.8 – 2.1; A260/A230 ratio should be similar indicating pure nucleic acids
3) Observe the absorbance spectrum of each RNA sample on the spectrophotometer used for quantification to ensure the peak is directly over 260nm.  If the peak is shifted to left (salt) or right (phenol) there is an organic contaminant present that needs to be removed prior to submission of sample(s)
4) RNA concentration should be 30-100ng/uL; if >100ng/uL please dilute prior to submission

  • PacBio Iso-Seq library preps, it is imperative the RNA is intact to ensure full transcript coverage.  Each sample is assayed on the Agilent Tapestation to assess the integrity of the RNA, NanoDrop to check purity and Qubit for accurate quantification.
  • We request a minimum of 12uL per sample as 3-4uL is needed for RNA QC.
  • If the RNA is suspended in something other than nuclease-free water, please include an aliquot when submitting your samples.
  • QC of RNA will occur within 1-3 business days of receipt.  QC results will be returned to the client with comments/suggestions.
  • Remaining template may be returned upon completion of project
  • Sample(s) will be disposed six months upon compleiton of library prep.

PacBio Iso-Seq RNA input range 300-500ng total RNA

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 abstracts/talks/publications.

To acknowledge our core, please use the following statement: “The author(s) thank the University of Wisconsin-Madison Biotechnology Center Gene Expression Center (Research Resource ID: RRID:SCR_017757) for providing Iso-Seq library preparation services”.

Please also let us know about your publication so we can list if on our website’s publication list!

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 Setup $1,450 $2,248
Per sample $170 $196

PacBio sequecning costs may be found here on the DNA Sequencing core’s Platforms page.

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

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.