PacBio IsoSeq

PacBio WGS PacBio AAV 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.		 $1,800.00
Revio 25M Flowcell
Run across a Full Flowcell. Library preparation separate.		 $2,000.00
PacBio IsoSeq Library Preparation
Includes QC and barcoding. One needed per sample. Sequencing separate.		 $300.00

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.

PacBio WGS

PacBio HiFi reads are ideal for WGS of complex, higher-order organisms and improve the likelihood of fully closed genomes. Library preparations are tailored to maximize read lengths for individual sample types, which helps you get the most out of your sequencing.

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PacBio AAV

As a proud PacBio-sponsored AAV sequencing facility, we are excited to offer library preparation and sequencing for both ssAAV and scAAV samples. The platform’s CCS-generated HiFi reads are a great way to validate vectors and track new mutations.

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Contact:
91 43rd Street, Ste. 250
Pittsburgh, PA 15201
(878) 227-4915

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Full Flowcell:

We offer full flowcell sequencing services for libraries that have been prepared outside of our lab. For each library, we will need to know the loading concentration, phiX spike-in, and run structure. Data is delivered as a complete run folder and can be demultiplexed upon request if a sample sheet is provided.

SARS-CoV-2 Sequencing:

Our SARS-CoV-2 Sequencing services include an assembly on the SARS-CoV-2 reference genome as a scaffold. These packages provide roughly 200Mbp of paired end reads (2×151 bp) as fastq files and the assembly in fasta format.

We require a minimum sample volume of 15 µL of extracted material (or 250µL for VTM samples) and recommend that only samples with Ct values below 35 are sent (because submissions are not measured for viral load before processing). We offer services for both purified RNA and samples in VTM media that require viral RNA extraction. Please see our SARS-CoV-2 Sequencing page for more detailed information.

Microbiome Sequencing:

Please see our Microbiome Sequencing page for package details, including distributables and sample requirements.

Nanopore Sequencing:

Our Oxford Nanopore sequencing packages provide reads equal to the length of input DNA, delivered as a fastq file.

For each sample, we require a minimum volume of 60 µL, and at least 40 ng/µL dsDNA quantified by Qubit or other dye-based method (100 ng/µL by Nanodrop). Variant calling add-on analysis requires an annotated reference file. DNA extraction services are available for only specific categories of microbes.

Please see our DNA Sequencing: Long Read Sequencing page for more detailed information about the package, variant calling analysis, and extraction requirements.

PacBio:

Our PacBio services deliver HiFi reads with an average read length of 8-12kb for standard offerings and up to 25kb for full flowcell runs. All package names are listed by the minimum number of >Q20 reads that will be delivered. Both assembly and annotation are included with all packages.

For each sample, a minimum volume of 60 µL and at least 35 ng/µL dsDNA (quantified by Qubit or other dye-based methods) are required. SeqCenter strongly recommends utilizing our HMW DNA Extraction service for best results; however, we will also accept samples that are shipped on dry ice to avoid DNA integrity issues caused by the shipping process.

Please see our PacBio Sequencing page for more detailed information about our services.

RNA Sequencing:

We offer both rRNA depletion RNA squencing and polyA-tail enrichment mRNA sequencing. Both provide stranded, paired end reads (2x 151bp) as fastq files.

For each sample, we require a minimum volume of 20µL and at least 50ng/µL RNA. We also recommend, but do not require, that samples be DNAse treated and have a RIN score greater than 6. We also offer Basic and Intermediate RNAseq analyses, which require an annotated reference sequence.

Please see our RNA Sequencing page for more detailed information about both our RNA sequencing and analysis packages.

Illumina Sequencing:

The Illumina sequencing packages produce paired end reads (2×151 bp) delivered as fastq files.

For each sample, we require a minimum volume of 30 µL and at least 10 ng/µL dsDNA quantified by Qubit or other dye-based method (20 ng/µL by Nanodrop). Variant calling add-on analysis requires an annotated reference file. DNA extraction services are available for only specific categories of microbes.

Please see our DNA Sequencing: Whole Genome Sequencing page for more detailed information about the package, variant calling analysis, and extraction requirements.

For additional information about variant calling and short read assemblies, please see our Bioinformatics page.

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