Genome Sequencing & Analysis Core Resource

PacBio RS

The PacBio RS sequencer is a third-generation sequencer that is able to sequence single molecules in real time without any signal amplification. This Single Molecule Real Time (SMRT™) technology is unique and novel as it makes possible the direct observation of DNA synthesis by a DNA polymerase. This offers four major advantages compared to other sequencing technoloy: 1) It allows for the observation of structural and cell type variation such as methylation; 2) Sequencing cost is significantly lower than other technologies; 3) Provides extremely long reads (up to 12kb) and unbiased sequences (i.e. balanced coverage and minimal GC-bias); 4) Fast results (less than a day).

Promoted by all the advantages listed above, this sequencing technology promises to revolutionize genomic analysis. It is well suited for several types of applications such as De Novo assembly of bacterial genomes, targeted sequencing, detection of base modifications, and finishing large genome sequencing. Sequencing is done on small chips called SMRT Cells. Each SMRT cell can produce 40Mb. Three sequencing modes are possible on this instrument:

• Standard sequencing of a 2kb insert library: this generates long continuous reads across thousands of individual polymerases in each SMRT Cell.
• Circular Consensus Sequencing (CSS) of a 250-500bp insert library: Because the insert is short and the library is circular, the same molecule can be sequenced multiple times (>3) and a high accuracy consensus is produced.
• Strobe Sequencing of a >6kb insert library: This mode increases physical coverage and read length by turning on an off the laser. This creates a series of long sequences that are spaced by known distances.

If you are a Duke employee, student or staff with a valid NetID, you can view the PacBio presentation given on October 8th by Jonas Korlach & Erich Jarvis

We offer both library preparation and sequence services. We can provide library preparation for short inserts (250bp to 3kb) and long inserts (3kb to 10kb).

Sample Requirements

The PacBio template preparation process does not utilize amplification techniques; therefore, input DNA quality will be directly reflected in the sequencing results. Any DNA damage (e.g., basic sites, nicks, interstrand crosslinks) or contaminants (e.g., single-stranded DNA, RNA, proteins, dyes, or salts, phenol) present in the input material will impair performance of the system.

Ensure that your DNA sample:

• Is double-stranded. Single-stranded DNA will not be made into a SMRTbell template and can interfere with quantitation and polymerase binding.
  
• Has undergone a minimum of freeze-thaw cycles.
  
• Has not been exposed to high T (>65°C for 1 h can cause a detectable decrease in sequence quality).
  
• Has not been exposed to pH extremes (<6 or >9).
  
• Does not contain insoluble material, and is not colored or cloudy.
  
• Does not contain RNA.
  
• Has not been exposed to intercalating fluorescent dyes or UV radiation.
  
• 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).
  
• Does not contain carryover contamination from the starting organism/tissue (like heme, humic acid, polyphenols).

Amounts Needed:

Recommended Extraction Kits:

PacBio recommends Qiagen gDNA extraction kits. Do not submit home-brew extractions. We need your protocol when you submit your sample. gDNA samples need an OD 260/280 ratio of approximatively 1.8 to 2. For PCR products, do not use gel cuts; our facility will clean up your pcr product for you.