SeqCenter will be closed Monday, May 27, 2024 in observance of Memorial Day. We will be unable to receive packages on this day. Two-week turnarounds will be offset 1 day by this closure.

PacBio IsoSeq

PacBio facilitates studies of novel isoforms. SMRT technology with CCS is uniquely able to provide true full-transcript sequencing with high per-base accuracy via HiFi reads. This combination enables the detection of all mRNA isoforms within the sample without requiring downstream assembly or inference.

As with all long-read RNA approaches, PacBio IsoSeq is best at providing a qualitative survey of a sample. This can be an excellent launching point for new discoveries. The data can also be supplemented by traditional Illumina RNA short-read sequencing as a cost-effective way to add complementary quantitative measurements.

For this pipeline, SeqCenter uses PacBio’s Iso-Seq library preparation protocol with the SMRTbell prep kit 3.0. This protocol works by converting poly-A tailed RNA to cDNA and ligating the SMRTbell sequencing adapters onto the cDNA transcripts. Library preparations are indexed to allow for multiplexing on any flowcell, and each order will need to include at least one flowcell, in addition to the preparation services.

In order to leverage the full advantages of this method and guarantee library preparation success, samples should have RIN scores greater than 7. We strongly recommend our customers evaluate their samples before submission, as all submitted samples will be processed.

Sample Requirements:

  • Minimum of 500 ng mRNA total
  • Maximum volume of 7µL
  • RIN >7 highly recommended
  • Anticipated length of transcript is required
  • Must be submitted on dry ice

What You Will Receive:

  • One fastq file for each sample
  • One bam file with run metric data
  • Sequencing stats summary
  • Materials and methods summary
  • Data shared via Box folder within 2 weeks

Packages & Pricing

Sequencing Package Price*
Sequel Ile 8M Flowcell
Run across a Full Flowcell. Library preparation separate.
Revio 25M Flowcell
Run across a Full Flowcell. Library preparation separate.
PacBio IsoSeq Library Preparation
Includes QC and barcoding. One needed per sample. Sequencing separate.

Due to the sensitive nature of RNA and IsoSeq protocols, we recommend an approach tailored to a project’s organism and experimental setup that is carried out in a uniform manner soon after harvesting.

For customer convenience, we offer RNA extraction services using the ZymoBIOMICS Quick-RNA Miniprep Kit. While broadly applicable, this protocol uses a bead bashing tube for consistent mechanical lysis and may not be the ideal extraction method for all organisms or for full-length RNA sequencing as it is prone to fragmentation.

This kit enables us to quickly isolate DNA-free RNA from cell pellets as well as homogenized tissue samples. We recommend sending cell pellets that contain at least 107-108 cells, in which the pellet is sizable and clearly visible in the tube. For tissue samples, we ask that you homogenize them as much as possible to enable successful extractions.

This extraction kit is compatible with solutions such as DNA/RNA Shield, RNAprotect, Allprotect, and RNAlater, but we request that customers include which specific product was used with their samples. More info on the ZymoBiomics kit can be found here.

Resources for IsoSeq Analysis

Unfortunately, we do not offer bioinformatic assistance for analyzing IsoSeq data at this time. PacBio does offer an IsoSeq pipeline for analysis that is freely available through their SMRT link platform that can further process the data into a consensus transcript sequence and predict open reading frames. More information on that platform and other PacBio tools are below.

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