Single-molecule, real-time (SMRT) sequencing is a third generation sequencing technology that generates long-read (ten thousand to a hundred thousand bases) length DNA and cDNA sequences. With the PacBio Sequel instrument, we are able to generate high-quality single-molecule consensus sequences of cDNA and amplicons <10 kbp long and contiguous sequence reads of 10-50 kbp length for long-read applications such as de novo whole-genome assembly and structural variant discovery. We are an HHMI-designated Pacific Biosciences (PacBio) Certified Service Provider offering services for library preparation as well as sample sequencing.
We provide library preparations and sequencing for:
*Maximum number of genomes per pool is dependent on expected genome size and desired coverage level.
We currently do not offer services for modified base analysis. Certain types of microbial modified bases can be determined from raw data. Please contact us for more information.
HHMI or UW-affiliated
External Academic or Nonprofit
Industrial or For Profit
|Template Prep Kit 1 Library Preparation and Multiplex Sample Prep Base Price||$429.98||$511.97||$646.17|
|BluePippin Size Selection with Additional DNA Damage Repair||$380.00||$452.46||$571.06|
|SageELF Size Selection||$493.60||$587.72||$741.79|
|Express Template Prep Kit Library Preparation with BluePippin Size Selection||$909.26||$1,082.63||$1,366.43|
|Iso-Seq Sample Preparation
|Sequencing Cost per Sequel SMRT Cell 1M v3 Run||$1,588.55||$1,891.45||$2,387.27|
|Sequencing Cost per Sequel SMRT Cell 1M v3 LR Run with 20 hr Movie||$2,160.23||$2,572.15||$3,246.4|
|*Price reductions may apply to pools of greater than 4 genomes.|
|Note: Prices effective 4/1/2019 and subject to change without notice.
Host institution and HHMI investigators have priority for the sequencing queue.
For Sequel SMRT cells, we provide the entire raw data folder as a tar.gz file, including the main raw data file in the subreads.bam format. We can also generate filtered subread sequences in FASTA or FASTQ format upon request. We include the following analysis services for specific sequencing project types:
|Sequencing project||Bioinformatics services|
|Bacterial genomic DNA||HGAP4
(one attempt at genome assembly using default parameters)
|Amplicons and HiFi Libraries||CCS2
(circular consensus sequence generation with loose or stringent quality filters)
(symmetric or asymmetric barcodes, standard 384 PacBio 16-bp barcode sequences)
|Iso-Seq||SMRT Link 6.0 Iso-Seq3 Pipeline
(standard analysis through cluster and polishing)
*If you would like us to demultiplex your custom barcodes, please specify this on the request form and include your barcode sequences.
Sequence data for your samples will be stored in PacBio's bam file format. You will be able to download these files from our Aspera server with a temporary account. Account details will be securely shared with you via Google Drive upon completion of your sequencing and analysis. Data and analysis from sequencing runs will be available through Aspera for 30 days and raw data will be backed up on our local storage for six months, after which it will be permanently deleted.
SMRT sequencing does not require template amplification. DNA quality will affect the resulting library and sequencing results. It is imperative that the DNA used is at high quality and sufficient quantity since there is no amplification step.
Follow these guidelines for sample preparation:
Other recommendations include:
If using AMPure beads, make sure there are no residual beads with the samples. This will inhibit any enzymes used in the subsequent process. It will especially affect polymerase binding critical for quality sequencing reads.
PacBio recommends using a high salt phenol/chloroform wash to remove polysaccharides from high molecular weight DNA. Polysaccharides are a common source of poor sequencing results, especially for fungal or plant samples. An alternative recommended kit is the PowerClean® DNA Clean-Up Kit to remove residual gDNA isolation reagents that may inhibit the polymerase binding essential for successful sequencing. The kit removes heme, polysaccharides, phenols, and dyes.
If you are barcoding amplicons before sending, PacBio’s recommended barcodes can be found here. All pooling must be performed prior to submission.
In addition to the sample preparation guidelines above, library preparations also require the following:
Please reference the tables below for DNA mass submission requirements. Ship DNA samples at 4°C. Do not submit DNA at room temperature as this may affect DNA quality. If using a UV absorbance quantitation method (i.e., Nanodrop or OD260), please submit more material as these methods tend to read up to 2x higher than fluorometric quantitation methods.
|Library Type/Insert Size||Amount of DNA Required||Input DNA sizing|
|Amplicon 100-750 bp||500 ng||100-750 bp|
|Amplicon 750 bp-10 kb||1 µg||750 bp-10 kb|
|HiFi Library 10-12 kb||3-5 µg||gDNA mode >20 kb|
|Genome 8-20 kb||3-5 µg||gDNA mode >20 kb|
|Large Insert Size-Selected Genomes (20 kb+)||5-8 µg||gDNA mode >50 kb|
|Multiplex Library Type/Insert Size||Amount of DNA Required|
|Multiplexed Amplicons 1-10 kb||1.5 µg total per pool|
|Multiplexed HiFi Libraries 10-12 kb||2 µg per sample for pools of 4 or more|
|Multiplexed Small Genomes 8-15 kb||2 µg per sample for pools of 4 or more|
For Iso-Seq library preps, submit the following amounts of RNA on dry ice. Do not submit RNA at room temperature. This may affect RNA quality. Submitted RNA should have a RIN score of at least 7.5
|RNA Type||Amount of RNA Required|
|Total RNA||10 µg|
|PolyA+ RNA||0.5-1 µg|
Follow the PacBio Standard Library prep protocol when constructing libraries for submission. Any deviation may result in poor sequencing run.
Make sure the final concentration is ~20-100 ng/µL at 15-200 µL volume (adjusting for required amount of DNA). Any less may result in a poor run or poor overall representation of template sequence.
Ship your samples to the following address using standard shipping protocols for DNA:
When your sequences are ready, you will be given a username and password for a temporary account on our local Aspera server where your data will be available for 30 days. To download your data, perform the following steps.
Data are grouped into folders by SMRT cell. Folders are named by the unique identifiers you supply for each sample. For each SMRT cell we provide a tarball (tar.gz archive) containing the entire output folder, including all information required for de novo assembly and any other secondary analysis pipeline from PacBio.
For sequencing projects where we provide additional secondary analyses, we will include a pdf report and the final output files with all FASTQ and FASTA files compressed by gzip.
SMRT Link, a GUI tool for analysis of raw PacBio reads, is available for download including the SMRT Analysis toolkit, which can perform de novo assembly and resequencing. PacBio also provides the latest pre-release versions of individual tools for command line use through Bioconda.