University of Washington PacBio Sequencing Services

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:

• Large-insert gDNA
• SMRTbell Template Preparation (8-20kb)
• SMRTbell Express Template Preparation (20kb+)
• High Fidelity (HiFi) Template Preparation (10-12kb)
• BAC-based DNA
• Purified PCR products
• Multiplex amplicons. Multiplex up to 16 amplicons.
• Multiplex small genomes (microbes). Multiplex up to 16 small genomes.*
• Transcriptome and Isoform sequencing with PacBio’s Iso-Seq protocol. This service includes cDNA generation from PolyA+ or total RNA and SMRTbell addition.

*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.

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:

1. If you are unsure which services you require, contact us for a consultation and a quote.
2. Fill out the sequencing request form or the Iso-Seq sequencing request form and include a purchase order (PO) number, fund code, or budget number. For external requests, we accept money order, VISA, and MasterCard payments.
3. Email the completed form to us at uwpacbio@uw.edu.
4. Prepare your samples and optionally prepare SMRTbell libraries for sequencing.
5. Wait for an estimate of cost form to be issued via email, sign it, and return to us.
6. Ship your samples as directed below.
7. Await email confirmation from us that your samples have arrived.
8. Await a sequencing report that will include details regarding how to download your sequence data.

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.

Sample Guidelines

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:

• The sample should be double stranded. Double-stranded DNA is required to make a SMRTbell template and single-stranded templates will interfere with quantitation and polymerase binding.
• Eluted in Qiagen Elution Buffer (EB) or similar buffered solution (10 mM Tris at neutral pH).
• Avoid freeze-thaw cycles.
• Do not expose to high temperatures (>65°C) for 1 hour. This may degrade the overall DNA quality and subsequent library prep quality.
• Has not been exposed to pH extremes (<6 or >9).
• Make sure it does not contain insoluble material and is not colored or cloudy.
• It does not contain RNA.
• It has not been exposed to intercalating fluorescent dyes or UV radiation.
• It does not contain chelating agents (e.g., EDTA), divalent metal cations (like Mg 2+), denaturants (like guanidinium salts, phenol), or detergents (like SDS, Triton-X100).
• It should not contain carryover contamination from the starting organism/tissue (e.g., heme, humic acid, polyphenols, polysaccharides, etc.).
• If submitting PCR products, use Qiagen kits for clean-up.

Other recommendations include:

• PacBio recommends ‘MasterPure Complete DNA and RNA Purification Kit’ (Lucigen) and Circulomics Nanobind CBB Big DNA Kit for High Molecular Weight (HMW) DNA extraction and purification. We also have had success with Qiagen or similar extraction kits for shorter fragment applications. Make sure to elute in 10mM Tris instead of water.
• Avoid vortexing where possible and use wide bore tips for pipetting. Higher concentrations of DNA help protect against shearing during routine handling.
• If performing AMPure bead washes, use very gentle (end-over-end) mixing and allow extra time for binding and elution. Elution may be assisted by short (~10 minute) incubations at 37°C.
• Tips for microbial DNA extraction:
  • Avoid incubation in complex or rich media.
  • Harvesting from several cultures rather than a single, high-density culture during early- to mid-logarithmic growth phase is preferred.
  • Extraction of small volumes is preferred over large volumes to avoid accumulating high concentrations of potentially inhibiting secondary components.
• Check for OD260/OD280 ratio between 1.8 and 2.0.
• Check for OD260/OD230 ratio between 2.0 and 2.2.
• Use low bind microcentrifuge tubes whenever possible.
• Run a standard or pulse field gel, BioAnalyzer, or TapeStation to visualize DNA quality. If sample looks degraded, re-extract the sample or plan for shorter read lengths.

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.

Recommended DNA clean-up

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.

Submitting Barcoded Pools

If you are barcoding amplicons before sending, PacBio’s recommended barcodes can be found here. All pooling must be performed prior to submission.

Library Guidelines

In addition to the sample preparation guidelines above, library preparations also require the following:

• Store in a stable environment; avoid freeze-thaw cycles, which will decrease library quality.
• Ship on gel packs or in dry ice with legible water/cold proof labels.
• Perform a BioAnalyzer or gel assay to see target library range and overall quality.

Sample Submission

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.

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

Library Submission

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.

Shipping Samples

Ship your samples to the following address using standard shipping protocols for DNA:

Downloading Data

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.

1. Install the Aspera browser plugin and restart your browser.
2. Navigate to the Aspera server.
3. Login with your given username and password.
4. Select all available files to download and click “Download”. From UW's campus we have experienced download speeds of ~40 Mbps.

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.

• Raw reads in PacBio's BAM (subreads.bam) format.
• All associated metadata files required for import of data into PacBio's SMRT Link software.
• Upon request, filtered subreads (≥50 bp, ≥75 read quality) in a compressed FASTQ.

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.