PacBio Sequencing

PacBio sequencing requires both a library preparation step and a sequencing step, which are priced separately. Described here is the end-result of both steps. If you click on the price link, please look for both library and SMRT cell prices. Consult with Core Specialists (Dr. McHenry or Dr. Lyons) to determine which procedures you need.

Sequencing of double stranded DNA as short fragments (250bp to ~ 2 Kbp) for high accuracy or long fragments up to ~20 Kbp for lower accuracy long read lengths. Reads are from the individual DNA fragments that were submitted with no amplification during processing.

 

PacBio Sequencing

Platform
Pacific Biosciences PacBio RSII
Category
Single Molecule Real-Time (SMRT) Sequencing
Primary Contact

Christina McHenry, PacBio Sequencing Specialist (cmchenry@umich.edu)

Typical Cost Click here to view internal pricing list!
Click here to view external pricing list!
Typical Output

Current results are approximately 40,000 to 90,000 reads at 3,000 to 15,000 bp average read length, depending on type and quality of sample and the specific sequencing method requested. Longest individual reads may reach 70,000 bp. Output can vary greatly for different samples due to type of sample (e.g. short templates run better than long ones) and individual characteristics of the DNA (damage or contaminants that inhibit the polymerase, etc.) but we generally assume that a good sample will yield 40,000 sequences at 10 kb average read length, for a total of 400 Mb of data output. Your result will probably be different!

How to Request

Sample Submission: Please see the 'Submit Samples' menu, above.

Sample Requirements

Amounts and concentrations of sample vary depending on the type of project and desired insert size for the templates. General ranges:

  • 250bp or 500bp without shearing = 250 ng dsDNA in 23 ul or less
  • 1 Kbp or 2 Kbp without shearing = 500 ng dsDNA in 23 ul or less
  • 5 Kbp without shearing = 2000 ng dsDNA in 39 ul or less

(Double the required amount above if shearing is needed.)

Genomic or other large DNA that requires shearing to 10-20 Kbp fragments for long template sequencing requires a starting amount of 10,000 ng dsDNA in 300 ul or less for two rounds of 150 ul shearing in Covaris gTubes.

Ask us if your sample does not easily fit into one of these categories. We can usually figure an amount that will work. Keep in mind that the smallest fragments in a mixture will tend to sequence preferentially so a mixture is best if the sizes are similar or if the bias for smaller sizes is acceptable.

Sample submission recommendations from Pacific Biosciences:

The Pacific Biosciences template preparation process does not utilize amplification techniques. As a result, input DNA quality will be directly reflected in sequencing results. Any DNA damage present in the input material (abasic sites, nicks, interstrand crosslinks, modified bases, etc.) will result in impaired performance in the system.

Therefore, ensure that your DNA sample:

  • Is double-stranded. Single-stranded DNA will not receive end-adapters in this template preparation process and can interfere with quantitation and polymerase binding. Qubit fluorometer or PicoGreen assay are both good for determining double-stranded DNA concentration. If you are quantitating with a Nanodrop or other spectrophotometer, please send excess as they tend to read other components besides dsDNA and overestimate the concentration.
  • 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), pH extremes (< 6 or > 9).
  • Has an OD260/280 ratio of approximately 1.8 to 2.0.
    An OD 260/230 of at least 2.0 is also recommended to avoid carbohydrate, phenol, and other contaminants.
  • Does not contain insoluble material.
  • Does not contain RNA contamination.
  • Has not been exposed to intercalating fluorescent dyes (e.g. ethidium bromide) or ultraviolet radiation (e.g. transilluminator.)
Typical Turnaround

Usually 3-4 weeks, unless we have received abnormally large numbers of samples submitted, or the sequencer has been down for maintenance or repair.

Critical Information

The sample will not be amplified so the quality of the DNA directly affects the sequencing performance. Concentration is best determined by a Life Technologies Qubit Fluorometer or a PicoGreen Assay, both of which only measure double stranded DNA. If you use a Nanodrop or similar spectrophotometer, please send extra sample as they tend to give higher readings due to detection of other components.

More Information

PacBio templates are produced by ligating single stranded end adapter loops to both ends of a double stranded DNA fragment. This creates a functionally circular template. The DNA polymerase will continue around the circle until the end of the observation period or until the polymerase becomes inactive. This allows short fragments to be read at high accuracy by creating a consensus sequence from multiple passes across the insert. Long fragments that are read only once will have an error rate around 15% but the long length allows scaffolding for contigs. Errors in PacBio sequencing tend to be random and so are easily correctable by generating consensus sequences. PacBio sequencing is tolerant of low complexity regions such as long repeats. Data is returned as FASTA and FASTQ files and metadata files for bioinformatics, with additional CCS (circular consensus sequence) files on request. Due to the unusually long reads and error rate, PacBio data is best analyzed by a Bioinformaticist using Pacific Biosciences SMRT Portal secondary analysis software (available from their website.) See link for information on bioinformatics analysis:

https://github.com/PacificBiosciences/Bioinformatics-Training/wiki

Researchers at the University of Michigan can contact the Director of the Bioinformatics Core to arrange for secondary analysis of PacBio data.