Element 101: Elevate or Adept Library Preparation for the AVITI™ System

Authors: Saeed Ahmed, Terrence Gelineau, Element Field Application Scientists

While Element Biosciences’ mission is to disrupt the genomics industry by democratizing sequencing, an important corollary is we aim to do that without disrupting your workflow. That’s why Element has created multiple options to streamline library prep for sequencing on the AVITI (Figure 1). Our workflow options include the Adept Library Compatibility Workflow – ideal for those libraries already prepared with third-party adapters, and the Elevate Library Prep – an alternative solution that integrates Element’s circularization sequences during the indexing PCR step. Whichever prep you choose – AVITI users always get to enjoy the high quality and reduced costs that Avidity Chemistry offers.

Figure 1: Overview comparison of the Adept and Elevate Workflows.

Adept Library Prep for Workflow Continuity

NGS users need a simple solution to take advantage of the innovations that the AVITI brings to the field of genomics. Linear NGS libraries prepared with third party indexes and adapters can be prepared for sequencing on the AVITI system using the Adept Library Compatibility Workflow. This simple workflow converts those libraries to libraries compatible with the AVITI.

  1. Prepare double stranded linear library from DNA or RNA.
  2. Anneal Adept splint oligos containing Element sequences to the library.
  3. Circularize the library.
  4. Digest linear DNA.
  5. Clean up circular library.
  6. Quantify the library.
  7. Sequence!

A regularly updated list of known compatible libraries can be found on the Compatible Kits for the Element Adept™ Library Compatibility Workflow webpage. Information for evaluating libraries not listed can also be found there, or you can contact Element Biosciences for assistance.

The splint oligo[CL1] contains sequences that are compatible with adapters found in many third-party library prep kits[DY2] . Some libraries may require amplification before circularization if there are 5’ or 3’ modifications or missing, additional, or mismatched bases on the library ends. Figure 2 demonstrates how Element’s Splint Oligos hybridize to the outer adapters of a third-party library’s fragment ends, where a perfect match-up is essential. Finally, Adept-specific PhiX is available for spike-in to facilitate error rate determination, increase nucleotide diversity in low diversity libraries, or for use in troubleshooting.

Figure 2: Adept library circularization shows how a splint oligo is annealed to linear, single-stranded DNA. The splint introduces Element sequences into the library, and library ends are ligated to the Element sequences.

Adoption of the Adept workflow requires minimal workflow changes and is ideal for labs wanting to benefit from sequencing by avidity without impacting already established library preparation protocols for samples that may need to be run on multiple sequencing platforms. The Adept workflow performs well with a wide variety of PCR-free and PCR-plus sequencing applications, including:

  • Human whole genome sequencing
  • Microbial whole genome sequencing
  • RNA sequencing
  • Single cell sequencing assays
  • Somatic and Germline sequencing
  • Whole exome sequencing

AVITI users with libraries who need custom sequencing primers will use the Adept compatibility workflow. Custom primers can be spiked into the AVITI Oligonucleotide Set that come with every sequencing cartridge. When a library structure does not allow for the primer spike-in method, replacement of read and/or index sequencing primers is possible using the Adept Custom Oligonucleotide Buffer Set.

Elevate Workflow for Seamless Circularization

The Elevate Library Prep solution is similarly easy to integrate into existing Next-Generation-Sequencing workflows. The advantage of using this workflow option is that the Elevate indexes and adaptors include features that improve the efficiency of the circularization step and reduce the requirement for PhiX during low-diversity sequencing. In addition, it eliminates any concerns around Adept compatibility. The Elevate Library Prep Kit contains two components, the Elevate Index and Adaptor Kit and the Elevate Library Circularization Kit.

The Elevate Index and Adapter Kit provides Element-specific adapter sequences and unique dual index pairs that are swapped into a compatible third-party linear DNA library prep kit. The “Element Index Pairs” are unique dual indexes (UDI) that are pre-paired in a 96-well plate format. UDIs minimize index misassignment rates due to library prep contamination and other factors, and the Elevate Indexes demonstrate excellent performance with high rates of index assignment at 97.6% (Figure 3).

Figure 3: Element indexes deliver uniform representation across multiplexed samples (A) Library concentration after linear library prep with Elevate indexes and adapters (CV = 0.035). (B) Index representation of 96 libraries with Elevate UDIs. The Y-axis shows the percentage of assigned indexes to each index pair on the X-axis.

The Elevate Library Circularization Kit contains circularization reagents and qPCR standards and primers specific to the Elevate workflow. This kit is used to prepare single-stranded circular DNA that can undergo rolling-circle-amplification on the AVITI. Both Adept and Elevate workflows give users access to the best-in-class Sequencing by Avidity NGS chemistry that Element Biosciences has pioneered.

The Elevate Library Prep solution is ideal for AVITI users who want to take full advantage of new features available with the upcoming Cloudbreak™ release while still using their favorite third-party kits. Element Bioscience’s Cloudbreak Chemistry takes workflow and ease-of-use to the forefront with many innovations. With Cloudbreak, DNA libraries prepared with Elevate indexes and adapters are circularized ­on-flow cell, with several advantages:

  • Reduced Hands-On Time
  • Minimal Sample Input
  • Less Room for User Error
  • Streamlined Quality Control Procedures

Cloudbreak Chemistry also enables AVITI to offer an indexing-first workflow. Indexing first enables early demultiplexing of your samples, allowing users to quickly understand sample representation in a pooled library. Understanding sample representation sooner creates the opportunity to make adjustments more quickly – reducing turn-around-times and giving users an edge in the fiercely competitive NGS space.

Conclusion

Whether you are aiming for that $200 Genome, or looking to achieve industry-leading quality scores, sequencing on the AVITI is right for anyone looking to utilize NGS technology for their research applications. With Cloudbreak Chemistry around the corner, run times, quality scores, and workflows will get even better than they already are today. AVITI users can be sure that Element Biosciences will continue to innovate and push the boundaries so that researchers can sequence more and better, enabling access to the genome for all.