PacBio Sequencing Services

Courtesy of Pacific Biosciences of California, Inc., Menlo Park, CA, USA

PacBio Sequel

The new Pacific Biosciences Sequel system builds upon their Single-Molecule, Real-Time (SMRT) technology and delivers higher throughput, increased scalability and lower sequencing costs compared to the PacBio RS II.

SMRT Sequencing

The Sequel system utilizes the DNA sample as a direct template for the sequencing reaction in order to sequence individual DNA molecules in real time. Ligation of hairpin adapters converts the double-stranded DNA molecules into a circular template that enables continuous sequencing of both the forward and reverse strands without the need for amplification.  The unique circular structure allows for continuous long reads of large-insert libraries or circular consensus sequencing of smaller insert sizes. Extra-long read lengths and high consensus accuracy make the Sequel a helpful tool for finding SNPs and sequencing through GC-rich areas of the genome.

Strengths and Applications

Library Preparation for Sequel

SMRTbell library preparation does not utilize amplification steps; the completed library molecules are used as direct templates for the sequencing process.  Due to this, the quality of the starting DNA has enormous influence on the success and quality of the sequencing results.  High-quality, high-molecular-weight genomic DNA is extremely important for obtaining long reads and optimal performance.

Sample Submission Requirements:

  • Gel image submitted with sample showing no signs of degradation.  For amplicon or cDNA samples, a Bioanalyzer trace can be submitted instead.
  • Nanodrop readings: 
    • 260/280: ratio should be around ~1.8; higher values are acceptable, low values indicate contamination/very low concentration.
    • 260/230: ratio should be ~2.0-2.2
  • Sample Concentration:
    • Use Qubit or PicoGreen for best results
    • Submit a minimum of 1.5 ug for <2kb libraries, 2.5 ug for 5 kb libraries, 15 ug for 20kb+
  • Volume and Buffers:
    • Max volume 130 uL
    • Avoid buffers using EDTA for DNA resuspension
  • Additonal Best Practices can be found here

Sequencing on the Sequel

The basic sequencing "unit" for the Sequel is the SMRTcell, analogous to a flowcell in Illumina sequencing.  SMRTcells do not have lanes, unlike flowcells, so each sample is sequenced on its own SMRTcell.  There are no "read lengths" like in Illumina sequencing; instead, you choose a length of time to continuously run the sequencing reaction, known as a "movie time".  The maximum movie time for the Sequel is 10 hours. 

There are two different methods of loading the SMRTcell: diffusion loading and Magbead loading.  Fragment size largely determines which loading strategy you will use.  For smaller fragment sizes (<7.5kb), diffusion loading is generally the recommended method.  In diffusion loading, SMRTbell library molecules with bound polymerase are immobilized and diffuse to the bottom of the ZMW wells after removal of excess polymerase and primer with cleanup beads.  For fragments >7.5kb, Magbead loading is recommended, which removes adapter dimers and short insert sizes to maximize read lengths.