BioMicroCenter:PacBio Library Preparation
|HOME --||SEQUENCING --||LIBRARY PREP --||HIGH-THROUGHPUT --||COMPUTING --||OTHER TECHNOLOGY|
Pacific Biosciences offers a broad variety of methodologies for preparing libraries for sequencing with a primary library prep kit called Express Prep Kit v2.0. Pricing includes quality control prior to prep. Large batches will be undertaken on a custom basis; please email email@example.com for more information.
dsDNA: Express Prep Kit v2.0
The BioMicro Center uses the Express Template Prep Kit v2.0 for dsDNA library preparation. This kit is recommended by PacBio for all libraries to be sequenced with Sequel Chemistry 3.0 for Sequel I. Single strand overhangs are removed, and DNA Damage repair is performed. End-Repair and A-tailing proceed before SMRTBell A/T Ligation, after which two rounds of SPRI cleans with PacBio approved beads clean the prepared library. Prepared libraries can be quantified via Qubit prior to polymerase binding. If a size selection is necessary, the size will be verified by Fragment Analyzer or by Femto Pulse for sizes larger than 6 kbp.
Typically, a good extraction of genomic DNA that has been stored appropriately should have fragment sizes larger than 100 kbp, even for bacteria. If sizes are smaller, it is likely that the DNA has been sheared somehow. If the average size is determined to be 50-60 kbp, the standard workflow is followed. If the average size is larger than 50-60kbp, the center has Covaris gTubes available for shearing to give a length slightly higher than 40 kbp. For larger average read lengths, other shearing methods can be explored on a case by case basis. After shearing and a potential size selection, the standard workflow proceeds.
The standard IsoSeq protocol uses polyA RNA as input. cDNA template is generated with the NEBNext® Single Cell/Low Input cDNA Synthesis and Amplification Module along with Template Switch Oligo from the PacBio Iso-Seq Express Oligo kit. Template is amplified with the NEBNext® High-Fidelity 2X PCR Master Mix and Forward PCR primer from the PacBio Iso-Seq Express Oligo kit. Enriching for longer transcripts requires longer elongation times for amplification. The normal protocol is then followed.