SMRT Sequencing FAQs

Single Molecule Sequencing is a Delicate Process

Here are some guidelines to help ensure successful library prep and sequencing:

Please ensure that the DNA...

•Is double-stranded.
Single-stranded DNA will not be made into a SMRTbell template in this template preparation process
and can interfere with quantitation and polymerase binding.

• Has undergone a minimum of freeze-thaw cycles.

• Has not been exposed to high temperatures (> 65ºC for 1 hour can
cause a detectable decrease in sequence quality). High temperatures will denature the double stranded ends, reducing the yield of blunt and a-tailed library ligations.

• Has not been exposed to pH extremes.

• Has an OD260/280 ratio of approximately 1.8 to 2.0.

• Does not contain insoluble material.

• Does not contain RNA.

• Has not been exposed to intercalating fluorescent dyes or ultraviolet radiation.

Does not contain chelating agents (e.g., EDTA) divalent metal cations (e.g., Mg2+),
denaturants (e.g., guanidinium salts, phenol), or detergents (e.g., SDS, Triton-X100).

• Does not contain carryover contamination from the starting organism/tissue
(e.g., heme, humid acid, polyphenols)

Input Material Required

We need at least 10 micrograms of genomic DNA. DNA is lost during the fragmentation and initial bead concentration steps-after which 5 micrograms of material is minimally is needed for the library prep. Presently we are requesting 10ug and ideally (if available) 15 ug per library prep to provide for thorough troubleshooting if necessary. Generating SMRTbell libraries involves many steps where yield is lost, including an exonucleation that reduces the amount of material significantly. No amplification goes into this process, so the more starting material the better.

The number of SMRT Cells to run will depend on your project aims. SMRT sequencing takes place on a SMRT cell and there are 8 per group – so the we run 8 SMRT Cells at a time (although we can of course accommodate less than 8 samples). Depending on the coverage required to achieve the aims of your sequencing project, some samples will require multiple cells. The current turn around time for library preparation and sequencing is three weeks to a month.

Data Analysis

Once the raw sequencing data has been generated it will be run through Pacific Biosciences proprietary software for evaluation of read length and quality. Detailed Read Metrics will be provided for all your samples and filtered data can be downloaded through Cornell's Large File Transfer Service, no account required.