While developing UltraQ kits, Element BiosciencesTM researchers determined that the leading contributors to error in next generation sequencing occur during the library preparation process (Q50+: Setting a New Standard for Sequencing Accuracy with Cloudbreak UltraQ). The two main sources of library-induced error are deamination damage from steps requiring heat, and mismatched base incorporation during the end repair process. UltraQ sequencing kits address these issues by removing damaged fragments prior to circularization and omitting end-repair errors by dark cycling through the regions most impacted.
For optimal performance from UltraQ sequencing kits, we recommend using the ElevateTM Mechanical Library Prep Kit (catalog # 830-00008) with Elevate Long UDI Adapter Kit Set A, (catalog # 830-00010).
While preparing libraries for UltraQ Sequencing, follow the protocol outlined in the Elevate Library Prep User Guide for the Mechanical Protocol with the following parameters:
- Fragment 200-1000ng high quality, intact gDNA with a Covaris ultrasonication shearer in a volume of 50 ul. Follow the manufacturer's protocol to target an average fragment size of 350 bp.
- During the Perform Size Selection step following the post-ligation cleanup, add 23ul beads (0.46X). Transfer 70ul of supernatant into a new well. To the supernatant, add 8ul beads (0.62X). The optimal final library target size for UltraQ is 450-550 bp.
- Do not perform the optional PCR amplification. Follow the PCR-free protocol for optimal results.
- Quantify final libraries by qPCR as described in the Elevate Mechanical Library Prep protocol using the primers and standards provided in the kit.
- For accurate size estimations, perform fork-collapse PCR on an aliquot of each library and analyze on a TapeStation or similar sizing instrument as described in our Knowledge Base article entitled “How can I improve accuracy in PCR-free library sizing?”.
Loading amounts and Sequencing parameters for Cloudbreak UltraQ.
- Consult the latest Cloudbreak Sequencing User Guide for loading recommendations based on your AVITITM model and AOS version. Loading amounts are generally lower to target the optimal range of 800M – 1B reads per run.
- Custom recipes such as HD Mode, PMG shift, Custom Filter Mask, Short and Long Insert are not compatible with Cloudbreak UltraQ.
- The individually addressable lane (IAL) feature is not available for Cloudbreak UltraQ runs.