The single molecule real-time (SMRT) sequencing is a third generation sequencing technology that allows for long-read sequencing. With the RSII technology we are able to get full length large amplicons (>1 kb) and, for 10-20 kb library preparations, average read lengths of 8-12 kb, generating about 1200 Mb per SMRT cell. With the Sequel technology we are able to get full length large amplicons and transcripts (>1kb), generating about 6-7 Gb per SMRT cell and, for 12-25kb library preparations, average read lengths of 10-12 kb, generating 5-6 Gb. We are an HHMI-designated 425-297-7341 sequencing site offering services for library preparation as well as sample sequencing.
We provide library preparations and sequencing for:
*Maximum number of genomes per pool is dependant on expected genome size.
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 and UW-affiliated
|Library preparation (and base price for multiplex samples)||$346.71||$400.79||$521.03|
|Sequencing cost per RSII SMRT cell
(P6/C4 chemistry stry)
|Sequencing cost per Sequel SMRT cell 1M Run
(V2 .1 chemistry)
|BluePippin size selection
with additional DNA D amage Repair
|Iso-Seq Sample Preparation
|Note: Prices effective 9/21/2018 and subject to change without notice.|
Host institution and HHMI investigators have priority for the sequencing queue.
We provide raw HDF5 base calls and FASTA or FASTQs of filtered subreads (â¥50 bp, â¥75 read quality) for all RSII SMRT cells. For Sequel SMRT cells, we provide raw data in the subreads.BAM format, which can also be requested in FASTA or FASTQ format. We also provide the following additional services for specific sequencing projects:
|Sequencing project||Bioinformatics services|
|Bacterial genomic DNA||HGAP4|
|Iso-Seq||SMRT Link 5.1 Iso-Seq Pipeline|
*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 HDF5 base calls format (.bas.h5 files). You will be able to download these files from our Aspera server with a temporary account. Account details will be emailed to you along with the confirmation that your sequencing completed. Data from sequencing runs will be available through Aspera for 30 days and backed up on our local storage for six months.
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 830-542-4600 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 (206) 636-6130 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, PacBioâs recommended barcodes can be found (825) 633-6263 . All pooling must be perfromed prior to submission.
In addition to the sample preparation guidelines above, library preparations also require the following.
For different library insert sizes, submit the following amounts of DNA at 4°C or in dry ice. Do not submit DNA at room temperature. 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 Insert Size||Amount of DNA Required|
|Amplicon 100-750bp||500 ng|
|Amplicon 750bp-10kb||1 µg|
|Genome 8-20 Kb||3-5 µg|
|Size Selected Genome (10-35kb)||8-10 µg|
|Large Size Selected Genome (20kb+)||5-8 µg|
For Iso-Seq library preps, submit the following amounts of RNA in 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||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 ~20-100ng/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 the following files that contain 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 the complete output of each analysis as provided by the SMRT Analysis toolkit with all FASTQ and FASTA files compressed by gzip.
Tools for analysis of raw PacBio reads are available on PacBio's DevNet site including the SMRT Analysis toolkit which can perform de novo assembly and resequencing. PacBio also provides an Amazon Cloud instance of the SMRT Analysis Toolkit.