The single molecule real-time (SMRT) sequencing is a third generation sequencing that allows for long-read sequencing. With the RSII technology, we are able to get an average of 4-6 Kb read length with 3-20 Kb library preparations, generating about 1200 Mb per SMRT cell. We are an HHMI-designated Pacific Biosciences (PacBio) sequencing site offering services for library preparation as well as sample sequencing.
We provide library preparations and sequencing for:
We currently do not offer services for modified base analysis.
|Service||HHMI and UW-affiliated||External
|Sequencing cost per SMRT cell
|BluePippin size selection
with additional DNA Damage Repair
|Prices effective 7/1/2016 and subject to change without notice.|
Host institution and HHMI investigators have priority for the sequencing queue.
We provide raw HDF5 base calls and FASTQs of filtered subreads (≥50 bp, ≥75 read quality) for all SMRT cells. We also provide the following additional services for specific sequencing projects.
|Sequencing project||Bioinformatics services|
|Bacterial genomic DNA||HGAP 2 assembly|
|PCR products||Reads of insert protocol|
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.
The following are recommendations from Pacific Biosciences for experimental design based on common applications of long-read technology.
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.
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|
|3-20 Kb||3-5 µg|
|Size Selected (10-35kb)||5-8 µg|
Barcode demultiplexing analysis is available for PCR amplicons. See PacBio's barcoding documentation for instructions. If you are using barcodes, specify this on the request form. We do not currently offer library barcoding services.
Follow the PacBio Standard Library prep protocol when constructing libraries for submission. Any deviation may result in poor sequencing run.
Due to low yields, 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 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.
The following are recommendations from Pacific Biosciences for improving large genome assemblies. See also the wiki page.
|Assembly approach||Software tool||Suggested PacBio coverage||Additional data sets||Genome size constraint|
|Hierarchical||SMRT Analysis - HGAP2||75-100X PacBio
|None||<150 MB provided sufficient compute power|
|50X short reads||Compute power and time|
|Scaffolding||AHA (SMRT Analysis)||10X PacBio
|High-confidence contigs||<200 MB; <20,000 contigs|
et al (2012)
(Note: beta release of PB Jelly 2 available for scaffolding too)
|High-confidence scaffolds||<4 GB
Compute power and time