Onyx® genotyping assays.

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Three genotyping assays. Many benefits.

Inscripta® offers three genotyping assays to assess the instrument run and analyze the composition of the Onyx cell library, both before and after phenotyping.
Assay1
The Control Edit Assay

A Sanger sequencing experiment managed by Inscripta. This assay detects the presence or absence of internal control edits in the genome in order to confirm the Onyx run resulted in successful edits.

Assay2
The Onyx Barcode Diversity Assay

A library preparation kit for Next Generation Sequencing using an Illumina platform. This assay detects the Onyx edit barcode, allowing users to derive the distribution of edits within the cell population.

Assay3
The Onyx Edit Identification Assay

A library preparation kit for Whole Genome Sequencing using an Illumina™ platform. This assay detects the edit sequence in the edited cell genome and can be run either on isolates (to identify the edit present in a particular clonal isolate) or pooled samples (to measure the overall edit fraction of cells).

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Easily characterize your Onyx Library.

Multiple factors will influence the genome engineering run, delivering a unique Onyx cell library. Understanding the library composition is necessary to plan the next phenotyping step.

Edit Identification Assay is used to: 

  • identify the edit present in an isolate of interest

Barcode Diversity Assay is used to: 

  • measure the designs observed in the Onyx cell library
  • compare the distribution of designs before and after the genome engineering run
  • help users plan their phenotyping experiments by providing information such as the percentage of the designed library observed in a given number of cells
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Identify the genotypes of interest.

Phenotyping is typically done in one of two ways, isolated or pooled. Determining which strategy to use depends on the goals of the experiment. Learn more about the phenotyping workflows utilized in Inscripta’s lab — read our phenotyping technical note.

Step 1: Isolate phenotyping

Onyx cell library clones are isolated and phenotyped individually. The number of clones screened to identify a certain number of edits is determined using the screener’s curve.

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Learn about Inscripta’s Lysine production strain engineering application which resulted in a 14,000-fold increase of lysine production in E. coli.

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Step 2: Identify the edits of interest

Edited cells with the phenotype of interest are then genotyped using the edit identification assay to determine the edit responsible for the phenotype.

Step 1: Pooled phenotyping

Pooled cells are grown in the presence of some selection pressure (drug, stress factor, etc.). Both positive and negative selection schemes can be employed to identify edits that respond to the agent being tested. The selector score is used to determine the number of cells to seed a selection experiment with, to ensure sufficient library representation.

Graph1

Download the application note for an example of a pooled phenotyping study using the Onyx Platform.

Download app note

Step 2: Identify the edits of interest

The cell population is then sequenced using the barcode diversity assay. This allows one to determine which edits are enriched or depleted and by extension deemed to provide a beneficial or deleterious contribution to cell growth.

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