How can I use TeloPrime to prepare full-length cDNA for long-read sequencing applications?

TeloPrime is compatible with library preparation protocols for analysis on Pacific Biosciences and Oxford Nanopore Technologies platforms.

TeloPrime V2 libraries have been successfully sequenced on PacBio Sequel® instruments. For further details see our TeloPrime Publications page, or contact support@lexogen.com.

Iso-Seq™

When TeloPrime cDNA is intended for Iso-Seq™ library preparation for sequencing on Sequel® instruments (Pacific Biosciences), additional adapters must be added to the ends of the cDNA to facilitate sequencing. For these applications we recommend the following:

• Use 1 – 2 µg of total RNA as input for TeloPrime cDNA generation.

• Use the highest possible quality of RNA.

• Purify the amplified TeloPrime cDNA after the endpoint PCR using Purification Beads recommended in the respective protocol from Pacific Biosciences (for further details see also Appendix D of the TeloPrime V2 User Guide).

TeloPrime V2 Iso-Seq libraries are best loaded for Sequel instruments using the diffusion loading method. Loading amounts around 6 pM have produced high outputs for TeloPrime V2 libraries. However, specific loading amounts may need to be adjusted according to the instrument and chemistry version in use.
Contact Pacific Biosciences for further advice regarding the Iso-Seq™ protocols and sequencing recommendations.
TeloPrime libraries have been successfully sequenced on MinION devices and are featured in several recent publications.

Oxford Nanopore Technologies

To prepare TeloPrime cDNA for downstream sequencing on Oxford Nanopore Techologies instruments, adapter sequences must first be added to the ends of the cDNA. In general, Oxford Nanopore Technologies kits for DNA library preparation (omitting any DNA fragmentation steps), or PCR barcoding can be used. Contact Oxford Nanopore Technologies for specific advice on the right kit for your chosen application.