Modeling homology assemblies with the combinatorial assembly tool

Homology cloning overview

Homology cloning allows you to join up to 10 DNA fragments into a single construct using homology regions, or complementary overhangs. This can be used to model: Yeast recombination cloning, Gibson, HiFi, Gateway cloning*, and/ or In-Fusion cloning**

*Gateway cloning: where all fragments already contain ATT regions

**In-Fusion cloning: fragments are modeled as having been PCR-amplified and include homology regions

Homology cloning set up

To open the combinatorial Assembly tool, see Combinatorial assembly tool: how to open and high through-put limitations

Within the combinatorial assembly set-up modal, fill out the following fields: 

• Name: Assembly Name
• Location: Project/ Folder the assembly will be stored in
• Number of fragment bins: Total number of backbone and inserts in the assembly
• Topology of construct: Circular or Linear
• Cloning method: Homology
• Ambiguous Construct Preferences (Homology specific field): In cases where the sequences you wish to assemble could create two distinct constructs, Benchling will create the construct that uses the larger/ smaller fragment in the first bin. This can apply when assembling two circular sequences, since each sequence produces two fragments

Click the Save button in the lower right corner of the assembly modal. This will open up a new assembly tab.

Edit name, project folder, topology of the construct, and cloning method specific parameters

All fields other than the Cloning method can be edited:

1. Within your assembly, select the gear icon located next to the assembly name at the top
2. In the assembly modal, you can make edits to the name, project folder, topology of the construct, and cloning method specific parameters
3. Click save

Configure bins

Bins are used to represent a fragment or group of fragments being used to assemble the final construct(s). Bins will automatically be set up based on the “Number of fragment bins” field in the assembly set-up modal.

Rename bins

Bins will automatically be named. The first bin will be named “Backbone” and all subsequent bins will be named “Insert (n)”. To rename a bin: 

1. Click on the current name
2. Enter the new name
3. Click out of that bin or hit enter to save the name

Add a bin

1. Near the top of the Assembly page, click the + next to the Bins & Spacers
2. Select Add new bin

Remove a bin

1. Click the gear icon located next to the assembly name at the top
2. Use the - near the “Number of fragment bins” to remove a bin

Rearrange bins

1. Click and hold the six dots in the upper right corner of each bin
2. Drag the bin to the new location and drop

Add fragments to bins

For Homology cloning, the entire sequence will be selected by default and cannot be changed. This is because Benchling scans each fragment to find homology regions that are compatible with its adjacent fragments, which may be:

• Anywhere in the fragment (doesn’t need to be at the 5’ or 3’ end)
• Different across two constructs at the same junction for the same fragment

You can add fragments using one of two standard methods:

Add fragments directly to bins

1. Click the + within the Bin
2. Select one of the following options to add a sequence: Open sequences, Search for sequences, or Add from worklist
3. The sequence(s) will populate automatically within the fragments table. In the table, select the desired orientation for each fragment.

Adding up to 1000 sequences to a bin at once

1. Click the + button of the bin you want to add the fragments to
2. Click Search for sequences
3. Click the three lines with dots
4. Within the dropdown, hover over the Results per page
5. Select the desired Results per page; options are 5, 10, 25, 50, 100, 500, 1000
6. Select all the sequences you would like to add from the single page

Add fragments via the Fragments table

Method 1

1. Double click into the Sequence cell
2. Begin typing the name and select the desired entity 
3. In the Bin column, select a Bin for each fragment. This will automatically update the Bins at the top of assembly tab
4. Choose the orientation of the fragment (forward or reverse)

Method 2

Copy and paste from a spreadsheet into the table

To learn about Fragment table tools, see Fragment and Construct table tools

Once the Fragment table has been filled out, the following grey-shaded fields will auto-populate:

• Length: length (in base pairs) of the fragment
• Status: tells you if the fragment has No errors, Contains Warning, or Contains error

Correct any status issues in the Fragments table

The status column will update automatically as you add fragments to the table. Each status can be defined as:

• Looks good: proceed to use fragment in assembly
• Contains Warning: can proceed to use fragment in assembly, but Benchling has identified a potential error
• Contains Error: can not use fragments in assembly until the error has been resolved

If the status is Contains Warning or Contains Error, an error message will appear within that cell. Prior to beginning to design your constructs, you will need to resolve any errors. Here are some tools to help:

Editing Fragments

Without leaving the assembly, you are able to edit the fragments being used via an Edit fragments pop-up modal.

Method 1

Edit one fragment

1. Right click on the row containing the fragment you want to edit
2. Select Edit fragment from the dropdown

Edit multiple fragments

1. Click on the first row containing a fragment you want to edit
2. Hold shift and click on the last row containing a fragment you want to edit. This should highlight all rows in between.
3. Right click on any highlighted row
4. Select Edit fragments from the dropdown
5. To move between fragments in the Edit fragments pop-up modal, click the ⋁ symbol next to the entity chip you want to open

Method 2

Edit one fragment

1. Click on the row containing the fragment you want to edit
2. Select Edit fragment located in the upper right corner of the table

Edit multiple fragments

1. Click on the first row containing a fragment you want to edit
2. Hold shift and click on the last row containing a fragment you want to edit. This should highlight all rows in between.
3. Select Edit fragments located in the upper right corner of the table
4. To move between fragments in the Edit fragments pop-up modal, click the ⋁ symbol next to the entity chip you want to open

Tools within the pop-up

• Entity blue chip: hover over this to see a small pop-up of the entity meta data or click on this to open the entity in another tab
• Orientation: use this to change the orientation to Forward or Reverse
• View (dropdown): change the view to one of the following- Sequence map, Plasmid map, Linear map
• Zoom (- to + slider): change the zoom setting of the sequence you are viewing
• Gear icon (upper right corner): change what features are present on the sequence you are viewing

Configure Construct Table

Once the status of everything in the Fragment table is No Errors or Warning, constructs can be assembled via the construct table. You can configure your constructs table using 2 methods:

Create constructs via “Auto-populate”

1. In the upper right hand corner of the Constructs table, click Auto-populate
   1. This will create constructs involving all possible combinations of fragments

Create constructs via Constructs table

Method 1

For every Bin, there will be a corresponding dropdown in the Construct table. Using the dropdown, select your desired sequence from each bin. If you don’t want to use a bin to create your construct, select Skip.

Method 2

Copy and paste from a spreadsheet into the Constructs table

To learn about Construct table tools, see Fragment and Construct table tools

Once you have filled out the Construct table, the following grey-shaded columns will auto-populate:

• Name: Assigned name of the new construct (can be changed later)
• Fragment (Backbone or Insert) start/ end: start and end coordinates of each fragment for the respective construct
  • This can vary by construct since Benchling detects junction-specific homology regions
• Overlap Length: length (in base pairs) of the fragment overlap at each junction
• Status: tells you if the construct has No errors, Contains Warning, or Contains error
  • If the status is Contains Warning or Contains error, an error message will appear within that cell. Prior to viewing and assembling your constructs, these errors will need to be resolved.

Visualize constructs

Once you have resolved all errors within the Constructs table, you are able to view your new constructs. Here are some tools to help users visualize constructs:

View constructs in pop-up modal

Method 1

View single construct

1. Right click on the row containing the construct you want to view
2. Select View construct from the dropdown

View selection of constructs

1. Click on the first construct you want to view
2. Hold shift
3. Click on the last construct you want to view. This should highlight all constructs in between.
4. Right click on any highlighted cell
5. Select View constructs from the dropdown

Method 2

View single construct

1. Click on the row containing the construct you want to view
2. Click the View construct button in the upper right of the table

View selection of constructs

1. Click on the first construct you want to view
2. Hold shift
3. Click on the last construct you want to view. This should highlight all constructs in between.
4. Click the View constructs button in the upper right of the table

Pop-up modal tools

• Sequence tab: view the sequence and features of your construct
  • Entity blue chip: hover over this to see a small pop-up of the entity meta data or click on this to open the entity in another tab
  • Orientation: use this to change the orientation to Forward or Reverse
  • View (dropdown): change the view to one of the following- Sequence map, Plasmid map, Linear map
  • Zoom (- to + slider): change the zoom setting of the sequence you are viewing

Constructs tab

You can view all constructs by clicking on the Constructs tab at the top of the assembly window. This tab will show a zoomed out view of your constructs and any associated primers.

Tools within the constructs tab

• Search bar: begin typing the name of a desired construct to quickly find it
• Left/ right arrows: toggle through the pages of constructs
• Results per page (lines with dots next to arrows): change the amount of results that appear on each page; options include– 10, 25, 50, or 100
• Blue entity chip: Click on this chip to open the construct entity in a new tab
• Diagonal arrows (next to blue chips): click these arrows to open the selected construct in the pop-up modal
• View (small blue button next to primers): this will open the pop-up modal to view the primers associated with your construct

Save assembly

To finalize your constructs:

1. Ensure all blocking errors are resolved, then click the Assemble button at the top right of the page.
2. Choose to create new sequences as needed:

• • Constructs (required)
  • Primers (optional, recommended if designed during assembly)
  • Fragments (optional, useful for saving fragments instead of PCR amplifying)

3. For each group of sequences (constructs, primers, fragments), select the following options:

• • Add to folder (required)
  • Add to worklist (optional)
    • Since worklists are limited to 500 entities, Benchling will split your new entities into separate worklists. The user can choose the standard name for the worklist. It will be auto-assigned a number in succession (ex. Name 1, Name 2, Name 3) 
  • Add to schema (optional; assigns a schema but does not register new sequences)

4. Access your finalized assembly from the project folder where it was saved.

Finalizing the assembly locks the assembly record, generates new DNA sequences for each construct, creates new primers where relevant, and populates the tables with references to these newly created sequence entities.