We offer a number of different sequencing services, including Pacific Biosciences single molecule sequencing. The Pacific Biosciences Real Time Sequencer can be used for:
- Whole genome sequencing of organisms with small genomes
- Full-length sequencing of >1kb PCR products
- Detecting epigenetic DNA modifications through observation of enzyme kinetics
- Rapid sequencing of a bacterial pathogen during an outbreak
- Transcript splice mapping
- Sequencing of repetitive, high GC or otherwise difficult templates
- SNP phasing
- De novo assembly of genomes and transcriptomes using long reads up to 6 kb
Sequencing Library Preparation
The Pacific Biosciences (PacBio) platform requires all DNA libraries to contain a common sequence on both ends, allowing the polymerase to bind and initiate sequencing. Large molecular weight DNA must first be sheared (the size may be from 250bp to 6kb depending on application). The PacBio adapters are short oligonucleotide strands which form a hairpin structure. These are ligated to the DNA fragments using standard molecular biology techniques. After ligation, the DNA library strands should be circular. If the double stranded portion of the library contains nicks or abasic sites, the polymerase will stop sequencing at that point. An exonuclease digestion is used to ensure all molecules are fully circular--the enzyme degrades nicked sample DNA, unligated sample DNA and unincorporated adapters.
The circular DNA library is then quantified on a fluorometer using a sensitive nucleic acid detection assay (SYBR-Green). This measurement is used to dilute the DNA to the correct molarity for sequencing. The completed libraries are annealed to the sequencing primer, and then incubated with the sequencing polymerase to allow binding to the template. The buffer does not contain key reaction components, so the polymerase does not synthesize the new template until later in the process.
The mass of input sample DNA needed varies with library size, as the sequencer performs most efficiently at a high library molarity (nano to pico molar range). DNA quality is a major factor, as the exonuclease step will degrade DNA which has been nicked by exposure to harsh chemicals, heat, acid, or trace amounts of nucleases. In addition, some chemical treatments or exposure to UV may oxidize, cross-link, or otherwise damage the DNA, leading to poor sequencing. Because there is no DNA amplification in this method, we advise 5 micrograms of starting DNA material.