Library preparation for Pacific Biosciences (PacBio) instruments differs in a number of ways from other next-gen approaches. The UMGC is deploying services and tools to support projects destined for sequencing on the PacBio platform. New methods and recommendations are continuously being developed by both PacBio and the UMGC, so please get in touch with us at firstname.lastname@example.org to discuss your application.
To read more about the underlying technology and instrumentation behind PacBio SMRT sequencing, review the page on the PacBio Sequel. http://www.pacb.com/products-and-services/pacbio-systems/sequel/
PacBio libraries are prepared with hairpin loops on each end, resulting in a molecule that is structurally linear, but topologically circular. Among the advantages of this approach is that, for moderately-sized libraries, multiple reads can be generated for a single template as the polymerase loops around the hairpin for a subsequent pass.
Size-selection is often used in PacBio sequencing due to a strong preferential bias in the technology for loading shorter fragments into the ZMWs. For projects looking for the longest possible reads, high-pass size-selection using the PippinHT (link to Sage Science PippinHT instrument page) is available. When a variety of size-ranges are of interest, the Sage ELF (link to Sage Science ELF instrument page) can be used to fractionate a single sample into multiple size bins, which are then sequenced on separate SMRTcells.
The success of PacBio sequencing is more sensitive to both the quantity and quality of the sample provided than other technologies. First, many PacBio library prep methodologies avoid PCR amplification, either due to a desire to explore epigenetic modifications or because of the limits of long-range PCR. In either case the original DNA base is carried through library prep and sequenced directly. Factors resulting in nicked, abasic, deaminated, or otherwise damaged DNA can impede both library prep and sequencing.
Additionally, longer sequencing reads require longer templates, which inherently increases the molecular weight and therefore the mass required for a given molarity.
In general, expect to submit more material of high quality than you might be used to. Visit the guidelines tab for more details.
Please see Pacific Biosciences under Instruments.
|PcBio. SMRTbell library preparation.|
|Includes fragmentation and Sage ELF gel cut||$282.75||$354.79|