Introduction
When preparing libraries for sequencing with Cloudbreak Freestyle™, the use of HPLC-purified oligos is recommended to increase the percentage of library molecules with full-length adapters. Imperfect synthesis of indexing primers can result in truncated adapter sequences, which occurs more frequently with less stringent forms of purification. While Cloudbreak Freestyle tolerates many modifications and truncations of library ends, significant truncations to the ends of the library can still impact circularization and sequencing, resulting in lower polony density than expected. Two mitigation strategies are available to improve sequencing results when non-HPLC purified oligos are used during library preparation or truncated library molecules are suspected.
End Polish
If libraries have already been prepared, conducting an end-polish PCR step after normal library preparation will “polish” fragments to ensure a higher percentage of full-length adapters. This process requires 4-5 cycles of PCR followed by a bead clean-up before final library QC. End-polished linear libraries can be loaded directly onto the AVITI™ and sequenced with Cloudbreak Freestyle.
Index Spike-In
The index spike-in method includes outer adapter primers along with indexing primers during the indexing PCR step of library preparation. This increases the full-length library percentage with minimal change to the library preparation workflow, requiring no additional PCR and no additional cleanup steps. The workflow can be optimized by adjusting the ratio of outer primer to index primer. A more detailed explanation of this method is available in the Truncated Library Molecules Technical Note (LT-00052). Linear libraries prepared with the index spike-in mitigation can be loaded directly onto the AVITI and sequenced with Cloudbreak Freestyle.