PacBio Sequel Sequencing


Overview

Since the Fall of 2016, when the UMGC acquired a PacBio Sequel instrument, we have been providing long-read sequencing to our clients. We currently offer the following PacBio sequencing applications:

  • De novo genome assembly –Our standard library prep offers an approximately 20 kb, size-selected library from high-quality genomic DNA. For particularly large or difficult genomes, we can work with you to develop ultra-long insert libraries that can maximize genome contiguity.
  • Genome re-sequencing –Re-sequencing with long PacBio reads can provide information that short-read sequencing technologies have missed. As little as 5X or 10X genome coverage of long reads can recover information on structural variants, including insertions, deletions, re-arrangements.
  • Multiplexed microbial sequencing –Recent improvements in protocols and tools for microbial sequencing on the Sequel platform have enabled increases in efficiency and reductions in cost. We can generally combine microbial genomes of approximately 5Mb in an 8-plex to produce single-digit contig assemblies.
  • RNA isoform sequencing – Isoform sequencing on the PacBio platform, termed IsoSeq, generates full-length, highly accurate consensus isoforms from high quality RNA. Recent experience with IsoSeq libraries shows we can expect >250,000 full-length reads per SMRT cell, generating 10,000-30,000 high quality transcripts.
  • De novo transcriptome sequencing and genome annotation – Sharing the same underlying methodology as IsoSeq, full-length transcript sequencing on the PacBio platform can serve as an invaluable resource for genome annotation projects, adding confidence to gene models and illuminating alternative transcripts.
  • Targeted sequencing –We are a PacBio focus site for targeted sequencing applications, especially target capture. We can work with you to plan, design, and execute target capture of long libraries (up to 10 kb) using probes from IDT, Nimblegen, or Agilent.
  • Epigenetic modifications –PacBio sequencing can natively detect certain types of epigenetic modifications in DNA with no additional modifications to sample preparation.
  • Long amplicon sequencing –A maximum of 96 amplicons up to 10 kb in size can be barcoded separately and multiplexed on a single SMRT cell. Expect around 200,000 reads-300,000 reads per SMRTCell using this method after demultiplexing.

SMRT Technology

Pac bio sequel

Whereas the Illumina platform detects unique fluorescence from clonally amplified DNA templates, the PacBio Sequel utilizes Single Molecule Real-Time (SMRT) sequencing to provide a direct real-time readout of each base from single DNA molecules. The SMRT technology uses one million zero-mode waveguides (ZMW) - microwells bound with a DNA polymerase - to capture signal from the incorporation of fluorescent-labeled nucleotides during DNA replication of SMRTbell templates. The detection system records a movie of light pulses that are converted to base calls to create a continuous long read from each ZMW.

Compared to the Illumina platform, that generates many millions of uniform-length short reads, the Sequel provides lower output in terms of read number, but yields a distribution of read lengths averaging tens of kb in length, with some reads ≥ 50 kb.

 

Additional information about the SMRT cell technology can be found on the PacBio website.

Output


Data output on the Sequel is highly dependent on the type of library and data is also more variable compared to other platforms. Given this, we do not guarantee any specific data output per SMRT Cell, but with the SMRT Cell 1M v3, we generally expect mean polymerase read lengths >10kb, with >250,000 reads under proper loading. This would result in total data output at about 4-6 Gb. For projects running multiple SMRT Cells, the loading concentration can often be titrated to achieve higher per-cell output.

Benefits


  • Generates long reads lengths averaging up to 10kb or more versus Illumina’s short read lengths of up to 300 bp.
  • Reads the same molecule multiple times for high consensus accuracy.
  • Does not rely on amplification during template preparation or sequencing, thus no PCR bias for more uniform genome coverage.
  • Direct read-out of base modifications, such as DNA methylation without chemical bisulfite conversion.
  • Resolves repetitive regions that are difficult for short read technologies.

Drawbacks


  • Produces many fewer independent reads compared to Illumina, hence not an ideal platform for read-counting applications such as RNA-Seq.
  • Far less output than instruments such as the Illumina NovaSeq, consequently making PacBio expensive for de novo sequencing of medium to large genomes.
  • Higher abundance of primary error rate compared to Illumina instruments (however, circular consensus sequencing can provide a very high-quality consensus sequence).

SMRTbell Library Creation


PacBio libraries are prepared with hairpin loops on each end that results in a molecule that is structurally linear, but topologically circular. For moderately-sized libraries, multiple reads can be generated for a single template as the polymerase loops around the hairpin for a subsequent pass to give a high-quality consensus sequence.

The UMGC provides high quality template preparation starting with rigorous sample QC to ensure only the highest quality DNA is used in SMRTbell creation. Since the SMRT technology sequences intact single DNA molecules, the quality and quantity of the DNA submitted for SMRTbell template preparation has direct influence on the length and quality of sequencing performance.

For projects looking for the longest possible reads, our PacBio service can also provide high-pass size-selection using the PippinHT. When a variety of size-ranges are of interest, the Sage ELF can be used to fractionate a single sample into multiple size bins, which are then sequenced on separate SMRT Cells.

Visit the guidelines tab for more details on sample requirements.

Questions?


To ensure our clients are getting the highest quality data and the longest reads possible, we undergo annual standardized PacBio certification, and also attend and speak at national PacBio User Group Meetings to stay on top of application updates and to exchange workflow best practices. Please contact us at next-gen@umn.edu for comprehensive support on experimental design.

 

PacBio Certification

 

Pricing

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Standard

Standard Long-read Service Scale Unit UMN External
SMRTbell <20 kb library creation.
Includes fragmentation and Sage ELF gel cut
1-any sample $345.97 $398.22
cDNA library creation for Iso-Seq. 1-any sample inquire inquire
Sequel SMRT Cell 1M v3.
10-hour movie
1-any cell $1295.58 $1533.22

 

Microbial Sequencing Service Coverage Scale UMN External

Tier 1

Includes PacBio library prep, SMRT sequencing, demultiplexing, and delivering of raw data for up to a 5 Mb genome. Coverage selected determines available multiplexing. Pricing is approximate per sample (please see Notes below)1.2
  75 16-plex $349 $380
  150 8-plex $458 $500
  150 16-plex $427 $475
  300 4-plex $730 $843
  300 8-plex $636 $726
  300 16-plex $585 $670
Tier 2
Includes tier 1 services + assembly and automated annotation. Sample cost is tier 1 + $75/sample for UMN and $100/sample for external.
Tier 3
Includes tier 2 services + Illumina polishing (Nextera library prep, MiSeq sequencing to 50X coverage, and bioinformatics). Sample cost is tier 1 + $330/sample for UMN and $391/sample for external
HMW DNA Extraction.
Optional add-on service using the Qiagen Genomic-tip for HMW DNA up to 150 kb. Pricing is per sample. N/A 1-any $86 $129
Bioinformatics.
Optional add-on informatic support. N/A hour $125 $144

 

Notes.

1. The pricing per sample is based on Statement of Work (SOW) that have a pre-set list of deliverables in terms of sample-plexity and number of SMRT cells (any variation from these pre-set criteria will alter the per-sample pricing). Since project needs are varied -- from the different strains that need to be sequenced to the sequencing depth required, we will provide a customized SOW for each project based on these variables, that may the alter the per sample price.

2. The coverage that mentioned in the table is the average coverage across a set of samples, with some samples receiving more than average and some less. PacBio recommends sequencing a maximum of 40 Mb on a Sequel 1M v3 SMRT cell.

 

Guidelines

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Submission

How to Order


Please contact next-gen@umn.edu for project specifications.

Once project details are finalized, complete appropriate submission form for submitting samples and email to next-gen@umn.edu.

 

Forms


PacBio Sample Submission Form

 

Samples can be dropped off at one of our laboratory locations.


 

1-202 Nils Hasselmo Hall (Minneapolis campus)

1-210 Cancer & Cardiovascular Research Building (Minneapolis campus)

20 Snyder Hall (St. Paul campus)

 

Shipping Instruction


Please give advance notice of submission date and time so staff can be prepared to receive samples. If shipping samples from outside the University of Minnesota, ship via express shipping carrier to the address below.

 

If shipping samples, the following address should be used.

Please send the tracking information to next-gen@umn.edu.

University of Minnesota Genomics Center

1475 Gortner Avenue

28 Snyder Hall

St. Paul, MN 55108

612-625-7736

Deliverables

Data Release


There are four options for transferring data from the UMGC to clients: 1) delivery to the Minnesota Supercomputing Institute’s (MSI) high-performance file system, 2) download from a secure website, 3) download with Globus, or 4) shipment on an external hard drive. Please indicate your data delivery preference when placing an order for sequencing.

1. MSI storage

Internal clients have their data released to MSI's Shared User Resource Facility Storage (SURFS). Delivered data will be located in the "data_delivery" folder in your group's folder on MSI's primary filesystem (home/GROUP/data_delivery/umgc). MSI does not charge for SURFS storage costs, but files expire and are removed one year after they've been delivered. Files should be copied to other MSI storage locations such as Tier2, Tier3, or your group's "shared" folder before they expire. 

2. Web download

Internal clients that opt-out of MSI storage and external clients can download their data from a secure website using either a web browser or a command-line download tool, complete instructions are provided in an email from the UMGC. The client’s data is available for download for 30 days, after which the data will be removed from the data download website and the client takes responsibility for storing the data.

3. Globus

Internal and External clients can use Globus to download their data. This is the recommended method for external clients to download large datasets.

4. Hard drive

External clients may have data shipped on a hard drive purchased by the UMGC and invoiced to the client at a cost of $250 per hard drive.

Data Recovery


The UMGC archives most customer data for a year and some datasets are retained for 5 years or more. If you need a dataset re-delivered email a request to next-gen@umn.edu to initiate data recovery. The UMGC does not provide any guarantee that data can be successfully recovered from the archive.