University of Washington PacBio Sequencing Services

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 Pacific Biosciences (PacBio) sequencing site 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+)
• BAC-based DNA
• Purified PCR products
• Multiplex amplicons. Multiplex up to 16 amplicons.
• Multiplex small genomes (microbes). Multiplex up to 8 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 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.

Host institution and HHMI investigators have priority for the sequencing queue.

We provide raw HDF5 base calls and FASTA or FASTQs of filtered subreads (≥50 bp, ≥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:

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 purchasing order (PO) number, fund code, or budget number. For external requests, we accept VISA and MasterCard payments.
3. Email the completed form to us at uwpacbio@uw.edu.
4. Prepare your samples and optionally prepare SMRTbell l ibraries for sequencing.
5. Ship your samples as directed below.
6. Await email confirmation from us that your samples hav e arrived.
7. 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 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.

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 template s will interfere with quantitation and polymerase binding.
• Eluted in Qiagen Elution buffer.
• Avoid freeze-thaw cycles.
• Do not expose to high temperatures (>65°C) for 1 ho ur. This may degrade the overall DNA quality and subsequent library prep quality.
• Has not been exposed to pH extremes (<6 or >9).< /li>
• Make sure it does not contain insoluble material and i s not colored or cloudy.
• It does not contain RNA.
• It has not been exposed to intercalating fluorescent d yes or UV radiation.
• It does not contain chelating agents (e.g., EDTA), div alent metal cations (like Mg 2+), denaturants (like guanidinium salts, p henol), or detergents (like SDS, Triton-X100).
• It should not contain carryover contamination from the starting organism/tissue (e.g., heme, humic acid, polyphenols, etc.).
• If submitting PCR products, use Qiagen kits for clean- up.

Other recommendations include:

• Use Qiagen extraction kits and elute in EB instead of water.
• Avoid vortexing where possible, if performing AMPure bead washes use very gentle (end-over-end) mixing and allow extra time for binding and elution.
• 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 larg e 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 gel or a BioAnalyzer to visualize DNA quality. If sample looks degraded, either re-extract the sample or clean up sample using a Qiagen kit or AMPure XP beads.

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 perfromed 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 in dry ice with legible water/cold proof labels.
• Perform a BioAnalyzer to see target library range and overall quality.

Sample Submission

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.

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

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 ~20-100ng/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.

Shipping Samples

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