What are the differences between the Trinity and Trinity Freestyle workflows for the AVITI and AVITI24™ systems?
Both Trinity and Trinity Freestyle deliver a streamlined hybrid capture workflow with on-flow cell capture and support a wide range of panel designs and sizes.
Trinity and Trinity Freestyle protocols and reagents are optimized for different library indexes and adapters and are not interchangeable. Trinity is compatible with Elevate™-style libraries, and Trinity Freestyle is compatible with P5/P7-style libraries. Trinity and Trinity Freestyle have different workflows, which are detailed in the Trinity Sequencing User Guide.
An overview of differences between the Trinity and Trinity Freestyle workflows is provided in the following table:
|
Workflow Section |
Trinity |
Trinity Freestyle |
|
Pre-hybridization Library Prep |
Use Elevate indexes/adapters |
Use P5 and P7 indexes/adapters |
|
Use library preps from Twist, IDT, and other vendors that are compatible with Elevate UDIs (unique dual indexes) |
Use Twist EF2.0 or FlexPrep library prep |
|
|
Multiplex up to 96 samples with Elevate UDIs (8 samples per hybridization reaction) |
Multiplex up to 96 samples or 96 FlexPrep pools with Twist UDI Primers for Trinity Freestyle |
|
|
Exome and custom panels supported |
Exome and custom panels supported |
|
|
Hybridization |
Standard and fast hybridization options available |
Fast hybridization only |
|
Run Setup |
Sequencing solution setup differs for standard and fast hybridization workflows (see user guides) |
Single sequencing solution protocol differs from Trinity (see user guide) |
|
Trinity PhiX Control |
Trinity Freestyle PhiX Control |
Protocols and reagents for Trinity and Trinity Freestyle are not interchangeable. Instructions, reagents, and reagent volumes can differ between workflows, so ensure you are accessing the appropriate protocol for your kit before starting.
Trinity sequencing requires Elevate indexes and adapters. The validated workflows for the Twist Bioscience and IDT exome panels provide an end-to-end solution from library preparation through hybridization and sequencing with Trinity. However, third-party libraries prepped with Elevate indexes and adapters can also be sequenced with Trinity.
Trinity supports custom panels. For more information about hybridization input and loading for custom panels, see How do I adjust loading for sequencing a custom panel with Trinity or Trinity Freestyle? For more information about plexity and pooling calculations, see How do I determine plexity and pool hybridization reactions for sequencing with Trinity or Trinity Freestyle?
Trinity sequencing includes both standard (overnight) and fast (1-2 hour) hybridization workflows. For standard hybridization conditions, see the validated Trinity exome workflows with standard hybridization:
For fast hybridization conditions, see the Trinity exome workflows with fast hybridization:
Hybridization conditions for custom panels can differ from the validated exome protocols due to differences in panel design, capture space, and other variables, and can require optimization.
The reagents and protocol required to create the Trinity sequencing solution differ for the standard hybridization and fast hybridization Trinity workflows. The following quick guides can be used as a reference by experienced users. For standard hybridization with Trinity, see:
For fast hybridization with Trinity, see:
Be sure to review and confirm run parameters before starting a Trinity sequencing run on AVITI or AVITI24. If you notice any errors in run setup after the priming step has completed, see the guidance for discarding a run setup for help restarting the process.
Trinity Freestyle expands the flexibility of Trinity to include P5/P7 library compatibility and expanded barcode access through the Twist EF2.0 and Twist FlexPrep library prep options. The expanded barcode access available through the Twist UDI Primers for Trinity Freestyle enables multiplexing of up to 96 samples.
The Trinity Freestyle workflow uses a fast (1 hour) hybridization during library preparation. For detailed prep and sequencing information, see the Twist Trinity Freestyle Hybridization Kit with Fast Hyb Workflow for Element AVITI Sequencing Protocol. You can find additional guidance in the Knowledge Base on custom panel loading, pooling, and modifying hybridization conditions for AT-rich panels.
Before starting, ensure that you are following the sequencing protocol for Trinity Freestyle, as the reagents and protocol differ from those in the Trinity standard hybridization workflow. The Twist Fast Hyb & Trinity Freestyle Run Setup Quick Guide can be used as a reference by experienced users.
If you are running Trinity Freestyle with Twist FlexPrep libraries, we recommend an 8-cycle PMG (polony map generation) shift. See the PMG shift section of the software documentation for help using the PMG shift feature.
Be sure to review and confirm run parameters before starting a sequencing run on AVITI or AVITI24. If you notice any errors in run set up after the priming step has completed, see the guidance for discarding a run setup for help restarting the process.