Considerations for Successful WGS PacBio Sequencing
The PacBio platform has some of the most stringent sample requirements of all of the DNA sequencing platforms we host. The extraction method and clean-up often have the greatest influence on sample quality in terms of purity and fragment length.
Expected Fragmentation by Sample Type
When ordering PacBio WGS, we strongly encourage you to include your sample type (e.g., metagenomic, cell culture, origin, etc.), organism, and extraction method. This information will help us fine-tune the fragmentation process to provide maximum read length and total output. Below are some examples of sample types, typical extraction methods we see, and average read length that might be expected.
|Typical Extraction Method
|Expected Read Length
|Bead-beating, physical lysis
|Bacterial cell culture
|Enzymatic, column-based, or HMW
|HMW kit, Nanobind Plant kit
|Up to 18kbp
PacBio and Amplicon Libraries
The PacBio SMRTbell prep 3.0 kit can support sequencing of amplicon libraries with modifications to our standard workflow to remove fragmentation. Due to skew for overrepresentation of smaller fragments and possible flowcell failure, it is strongly recommended that library lengths differ by no more than 10%.
To ensure proper processing of amplicon libraries, please note that the sample is an amplicon library and that fragmentation should not be performed. For additional information, please contact us.
PacBio Sequencing and Modified Bases
PacBio’s SMRT technology is capable of capturing raw methylation kinetics that can later be used to predict 4mC or 6mA base modifications. In order to store this data, the sample must be flagged for this option in advance of sequencing, so customers must indicate that the optional data should be stored for their samples when placing an order. Post-sequencing, predictions can be made in relation to an assembly but cannot be placed on individual reads. For additional information about SeqCenter’s add-on services for methylation calling, please see the PacBio Long Read portion of our Assembly and Annotation page.