The Pacific Biosciences Sequel (PacBio) is the second generation sequencer from PacBio and generates up to 7x more reads than its predecessor, the RSII.  Get full length iso-form data without the need for assembly.  We will be adding on the Iso-seq application soon. 

PacBio

Sample requirements for PacBio sequencing

PacBio library preparation does not include amplification techniques, and thus utilizes native DNA templates for sequencing.  Therefore, the quality of the input DNA will be directly reflected in the sequencing results, as any irreversible damage present in the DNA will result in impaired performance in the system.  Both high quality, high molecular weight genomic DNA is imperative for obtaining long read lengths and optimal sequencing performance.

Important measures impacting DNA quality

  • DNA must be double stranded; single-stranded DNA cannot be used to generate sequencing template.
  • Has not undergone multiple freeze-thaw cycles, as that can lead to DNA damage.
  • Has not been exposed to high temperatures (>65C for 1 hour) or pH extremes (<6 or >9).
  • Has an OD260/OD280 ratio of 1.8 to 2.0.
  • Has an OD260/OD230 ratio of ~2.0
  • Does not contain insoluble material, carryover contamination from the organism/tissue.
  • Does not contain RNA contamination
  • Has not been exposed to intercalating dyes, UV radiation, or ethidium bromide.
  • Does not contain denaturants or detergents

DNA sample quality assessment

A DNA quality check is imperative before submitting sample for PacBio sequencing and/or starting the library preparation.  The following recommendations for determining DNA integrity, purity, and concentration are highly recommended.

  • Genomic DNA integrity can be assessed by pulse field gel electrophoresis; the presence of one predominant high molecular weight band with no degradation is optimal. Amplicon or cDNA samples can be run on the bioanalyzer as an alternative to gels.

Purity of your DNA sample

Readings of both A260/A280 and A260/230 ratios on a Nanodrop instrument need to be obtained to make a determination of purity.

  • The ratio of absorbance at 260 nm and 280 nm is used to assess purity of DNA.  A ratio of ~1.8 is generally accepted as pure for DNA.
  • The 260/230 ratios provide a secondary measurement of DNA purity to assess the quality of the extraction.  Expected 260/230 values are commonly in the range of 2.0-2.2.  Abnormal 260/230 values may indicate a problem with the extraction procedure.

Concentration of DNA sample

  • For PacBio library preparation, it is critical to determine the concentration of the double-stranded DNA present in the sample.  Spectrophotmetric assays do not distinguish between different types of nucleotides, therefore it is highly recommended to use an intercalating dye on a fluorometer like the PicoGreen assay, or a Qubit for quantitation purposes.

 

Contact Aaron Trippe or Brent Kronmiller to discuss your project and to find out how the Sequel’s technology can work for you.  Pacific Biosciences has been steadily introducing more streamlined workflows for the Sequel that could impact pricing. Inquire about the latest pricing when discussing your project.