How do I determine plexity and pool hybridization reactions for sequencing with Trinity™ or Trinity Freestyle™?

Written by Element Biosciences | Sep 14, 2026, 9:31:59 PM

Question

How do I determine plexity and pool hybridization reactions for sequencing with Trinity or Trinity Freestyle on the AVITI™ or AVITI24™ systems?

Answer

You can estimate plexity by dividing your desired per-sample coverage by the expected flow cell output, taking expected losses into account.

All Trinity workflows provide instructions for diluting the hybridization reaction before preparing the final sequencing solution. The correct proportion of the diluted reaction is determined by your protocol and any customizations you choose to make.

For some small custom panels, you might have used multiple hybridization reactions to achieve your desired total sequencing output. In this case, pool all of the hybridization reactions into the same final sequencing solution before loading the library well.

Summary

Plexity and pooling can be adjusted for Trinity and Trinity Freestyle to meet loading requirements.

Estimate plexity using the ratio of per-sample coverage to expected flow cell output, taking expected losses into account
Use multiple hybridization reactions to achieve your desired sequencing output if needed
Pool all reactions in the same final sequencing solution before loading the library well

Considerations

Plexity

To determine plexity, first decide how many paired-end reads are needed per sample, accounting for read length in the desired coverage, expected on-target rate, duplication rate, and any other expected losses. You can contact your Field Applications Scientist or Support for help with this calculation.

Divide the total read requirement per sample by the expected flow cell output (such as 800M paired-end reads for AVITI or 1.2B paired-end reads for AVITI24) to estimate how many samples can be multiplexed. Check your library preparation guidelines for any limitations regarding plexity per hybridization reaction or pool.

For help estimating the total loading concentration for your custom panel, see How do I adjust loading for sequencing a custom panel with Trinity or Trinity Freestyle?

Pooling

For some small custom panels, you might have used multiple hybridization reactions to achieve your target sequencing output. In this case, after diluting each reaction, pool all of the hybridization reactions into the same final sequencing solution before loading it into the library well.

An example of this is provided in the following tables for a custom panel that required two hybridization reactions to be combined in the final sequencing solution:

Example Sequencing Solutions (Combining Two Hybridization Reactions)

Trinity Standard Hybridization		Trinity Fast Hybridization		Trinity Freestyle Hybridization
Component	Volume	Component	Volume	Component	Volume
Library Loading Buffer	1728 µl	Trinity Fast Hyb Loading Buffer	1728 µl	Trinity Fast Hyb Loading Buffer	1764 µl
Trinity Sequencing Reagent	72 µl	Trinity Sequencing Reagent	72 µl	Trinity Freestyle Sequencing Reagent	36 µl
Diluted Hybridization Reaction 1	200 µl	Diluted Hybridization Reaction 1	200 µl	Diluted Hybridization Reaction 1	200 µl
Diluted Hybridization Reaction 2	200 µl	Diluted Hybridization Reaction 2	200 µl	Diluted Hybridization Reaction 2	200 µl
Total	2200 µl	Total	2200 µl	Total	2200 µl

In the example tables, two diluted hybridization reactions are combined in their entirety in each of the workflows. The Loading Buffer amount is reduced to accommodate the additional volume of both reactions. You can further adjust the volume of the Loading Buffer in your workflow to accommodate any volume or combination of diluted hybridization reactions, as long as the final volume of the sequencing solution is 2200 µl.

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