What are the differences between the Trinity™ and Trinity Freestyle™ workflows for AVITI™?

Question

What are the differences between the Trinity and Trinity Freestyle workflows for the AVITI and AVITI24™ systems?

Answer

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.

Summary

An overview of differences between the Trinity and Trinity Freestyle workflows is provided in the following table:

Considerations

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

Pre-Hybridization Library Prep

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?

Hybridization Conditions

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:

• Twist for Element Exome 2.0+ Library Preparation and Standard Hybridization with Trinity Sequencing Workflow
• xGen Exome Sequencing Kit Trinity for Element AVITI Family of Systems

For fast hybridization conditions, see the Trinity exome workflows with fast hybridization:

• Twist for Element Exome 2.0+ Library Preparation and Fast Hybridization with Trinity Sequencing Workflow
• Trinity Workflow with IDT xGen Exome Kit and 1–2 hour 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.

Run Setup

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:

• xGen Exome Hybridization & Trinity Run Setup Quick Guide
• Twist Exome Hybridization & Trinity Run Setup Quick Guide

For fast hybridization with Trinity, see:

• Twist Exome Fast Hybridization & Trinity Run Setup Quick Guide

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

Pre-hybridization Library Prep

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.

Hybridization Conditions

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.

Run Setup

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.