Genomics Core Facility

Pacific Biosciences RSII DNA Sequencer (operated by Core Staff)

 

• This instrument was revolutionary when introduced in being a single molecule sequencer capable to long DNA sequence reads (may exceed 40Kb). This made the SRII the first long read sequencer capable of bridging large repetitive genome regions. Since its introduction a number of applications have been developed
  • Whole genome sequencing is its most significant application
  • Targeted resequencing
  • RNA sequencing
  • Epigenetics
• For sequencing, DNA fragments of the desired length are ligated to SMRT Bell adaptors which both create a circular template and provide polymerase binding sites. Sequencing can then proceed based on the tethering of polymerase-bound DNA templates within minute pores within a SMRT cell. The incorporation of single nucleotides is then detected using optics to provide real time sequencing of individual template molecules, one for each pore (zero mode waveguide or ZMW).  For overview of the RSII workflow see the PacBio web site.
• Sequencing is done using SMRT cells which contain 150,000 ZMWs. This results in a yield of about 30,000 to 60,000 individual sequence reads per cell.

• The instrument is capable of running up to 16 SMRT cells in a single run. SMRT cells are purchased in packages of 8.
• Each SMRT cell is interrogated by creating a movie of ZMWs as incorporation occurs in real time. The movies are interpreted as base calls using software known as SMRT Analysis resulting is FASTQ data files for analysis. Movies can be from 30 mins to 6 hrs, the longer the movie the greater the average read length. For an overview of RSII technology see the PacBio web site.
• Average read length for the RSII is 10 to 20Kb. This can be altered by size selecting longer insert libraries using the Blue Pippin instrument at the Genomics Core.
• Typical library lengths are 2Kb or less, 10Kb, 20Kb and longer. This may require the isolation of higher molecular weight DNA and shearing using mechanical means such as a syringe or G-tube.

Samples

• Samples are run by Core staff on a per sample fee basis.
• For prices of sample prep (required) and sequencing per SMRT cell please contact the Genomics Core.
• Sample submission utilizes the same forms as Illumina sequencing. For submission details please contact the Genomics Core.