DNA and RNA sequence overview

Benchling’s DNA and RNA sequence features offer a modern, collaborative way to manage, analyze, and design genetic constructs. Instead of relying on disparate software and files, you can centralize your RNA sequences in Benchling, creating a single source of truth for research. This not only improves organization but also facilitates collaboration and ensures data integrity.

Benchling's cloud-based nature allows for seamless collaboration. Teams can work on the same sequences in real-time, reducing redundancy and improving hand-offs. The platform helps standardize sequence analysis and registration across an entire organization.

This article provides comprehensive guidance on managing DNA and RNA sequences within Benchling, covering importing, viewing, editing, exporting, bulk operations, sharing, and locking sequences.

Supported sequence file formats

Benchling supports many common and proprietary file formats. Using a supported file format means that Benchling will import the sequence, annotations, and comments. 

We recommend GenBank for processed sequences, ab1 for trace data, and then FASTA and raw bases for raw sequence files over any other file types. The list below provides additional non-principally supported file types that can still be imported into Benchling.  

If you encounter an import error, ensure your file extensions are formatted correctly. If you encounter an import error due to an unsupported file format, try saving or exporting your file in GenBank or FASTA format. Benchling does not currently support .pdf, .doc, or .docx file types for sequence import. To import sequences from these files, you can manually copy and paste the bases into Benchling.

Certain formats support additional capabilities: 

• Multi-file types – support multiple sequences that are automatically split by Benchling when imported at once
• Trace file types – results from Sanger sequencing and contain chromatogram traces and quality data when imported as an alignment

Fields on GenBank files

GenBank files come with fields of different categories and sub-categories which are respected when loaded into Benchling.

There are:

1. Fields that characterize the whole sequence like accession, notes, etc  

• When imported into Benchling these will populate as custom fields on the metadata page for the whole sequence 

2. Features that characterize regions of the sequence

• Features of type CDS will become translations on the sequence. In GenBank these have trans_table properties that set the genetic code. Translations can have custom fields
• Features of other types will become annotations on the sequence. Annotations can also have custom fields

Create DNA and RNA sequences

You can manually type in a sequence or import a sequence from a file on your computer, a database, or a genomic locus. 

To create a sequence in Benchling:

1. Click the Global Create in the left sidebar
2. Hover over DNA/RNA Sequence, then click New DNA/RNA Sequence
3. This opens the Create DNA / RNA Sequence modal, which displays four ways to create a sequence, select the sequence input method by clicking:
   • Create New to manually input a sequence by pasting the bases into the text bar
   • Upload Files to upload a sequence file from your computer
   • Import From Database to import common sequences from external databases
   • Select Chromosomal Region to import from a genomic locus

The sections below discuss the steps for each method of sequence creation. 

Create new

1. Click into the Create New tab
2. On this page define the sequence name, set the nucleotide type as DNA or RNA based on the sequence type you’re creating and select a folder to save it to
3. In the Bases text box, paste the sequence from your clipboard (recommended) or type it manually
4. After ensuring all sequence information is complete, click Create

Upload file

1. Click into the Upload Files tab
2. Select the folder to save your sequence to
3. Click Choose a file and select the file(s) from your computer or drag and drop the file(s) into the box
4. Update the file location, sequence topology, or tags associated with the file, as needed
5. Click Open Sequence to open the file, or click Close to close the modal

Import from database

1. To import sequences from external databases, click into the Import From Database tab
2. Use the Sequence text box to enter the name of the gene before clicking Search
   • You can specify different IDs depending on where you want to import the sequence from
3. If multiple matches are found, use the Genome dropdown to select the species for the sequence you want to import, then click Search
4. Define the attributes of the sequence by editing the name so it displays as you want it to appear in Benchling
5. Select how you want to import the sequence as either a Genomic Sequence or DNA or RNA
6. Define a folder for storage, nucleotide type and optionally a schema if you wish to register the sequence 

Benchling supports the following inputs for pulling active DNA sequences from external databases:

• Addgene URL
• BioBrick ID
• Ensembl ID
• Gene name or symbol in Ensembl
• Joint BioEnergy Institute (JBEI) ID
• NCBI Accession Number

Tip: If searching by a gene name returns a result in Ensembl, you can update or select multiple transcripts.

Create DNA sequences by selecting chromosomal region

Unlike the options listed above that are shared sequence creation methods between DNA and RNA sequences, selecting a chromosomal region is an option limited to creating a new DNA sequence.

1. Click into the Select Chromosomal Region tab
2. Use the Genome text box to enter your search terms to find active genomes, then click Search
   • You can search by common name, species, or version
   • We support the most recent Ensembl genome versions
     • In limited cases, we support prior versions
3. Use the dropdown to select the chromosome of interest and the start and end loci of the target sequences
4. Define a folder for storage, nucleotide type and optionally a schema if you wish to register the sequence
5. Click Import

Create sequences using a Registration table

If your experimental protocol results in new sequences that you want associated with the experimental context in the Notebook, you can create and register the sequence using a Registration table. 

1. From the Insert menu in the toolbar, select Registration table
2. In the Insert registration table modal that opens, choose whether you want to Create new entities or Register existing entities, depending on whether you want to import new entities or register existing, unregistered ones
3. Use the dropdown to select the Registry schema for the entity. A table preview will display for you to review before inserting into the entry. Use this to confirm your selection, then click Insert
   • If you select a DNA or RNA sequence, a Bases column populates next to the Entity Name column
4. Enter information about the entity into a row of the Registration table. Each row corresponds to one entity
5. Click Submit to register the entities
   • Note: The columns of the table are defined in the Registry configuration

Create sequences using spreadsheet import

Benchling allows for the efficient creation and registration of many DNA or RNA sequences at once. This feature is particularly useful for larger data sets. In some cases you may want to bring in Registry metadata and fields as you create the sequence entities. This can be done through a spreadsheet upload then “reimporting” can be used to bring in the sequence annotations in bulk. 

Prepare a spreadsheet for DNA or RNA import

For a successful import, the spreadsheet should be formatted as outlined below:

• Name (Required) - The values in the column need to exactly match the name of your DNA or RNA sequence files from Snapgene, Geneious, or other software
• Bases (Required) - If you are hoping to import annotations and other sequence features, keep this blank for now, the sequence maps can be reimported in a later step
• Schema fields (Optional) - Fields that have been configured on the schema to capture entity metadata as part of a data model ex. Vendor, Antibiotic resistance, etc.

1. Navigate to the Registry on the navigation bar
2. Click the + and then click Import Entities
3. Select the schema of the entities you're importing and use the check box to indicate your DNA or RNA is circular, if necessary, then click Next
   • Note: If you're using the Inventory application and would like to simultaneously create a container and deposit it into a Location (i.e. Box), then select Transfer entities to new or existing containers and designate the container schema
4. Now add your spreadsheet, you can either import the full spreadsheet or copy and paste the fields from your spreadsheet template into the raw text box. Please ensure that you include the column names.
5. Once this is done, click Next
6. In the next menu, ensure that the column names pulled from your spreadsheet match the column type fields. Once they're matched click Next
7. Benchling will now check for errors, if no there are no errors, click Import
   • If there are errors, you will need to resolve them before you can continue
8. Your entities will now be viewable in the Registry

Reimport sequences

Reimporting is useful in cases where you are trying to bring in sequence features (such as annotations, translations, etc.) for sequences that have already been imported and registered in Benchling. 

1. Within the Registry click the local create button and select Reimport DNA/RNA Sequences
2. In this menu, you can now drag and drop in the sequence files, the file name must be identical to the entity name in Benchling to be matched with the entities you created earlier
3. Once you add all the files, select Reimport
   • Each sequence file should contain only one sequence per file, as Benchling will generate an error if multiple sequences are found in a single sequence file

Now that you have imported your sequences you can analyze various information about them. 

View DNA and RNA sequences and change display features

When viewing a sequence in Benchling, you can highlight any section of RNA by clicking and dragging your mouse over that section. The bottom status bar will populate with information about that sequence. You can view the following properties:

• Bases, or the length of the entire sequence including parts that are not highlighted
• Start base
• End base
• Length of the highlighted section
• Melting temperature (Tm)
• GC content

Edit and personalize your sequence view

You may want to have different sequence tabs open at once to compare and contrast different views or easily gather data from one tab into another.

Toggle on or off the split workspace by clicking Split Workspace on the bottom right corner. You can also drag the tabs of the plasmid map around in your preferred order.

Control sequence map visual settings

Click the gear symbol on the upper right corner to toggle on and off different features of the plasmid map.

• Annotations - determines whether annotations linked to the sequence will be displayed on the sequence map
• Primers - determines whether primers linked to the sequence will be displayed on the sequence map
• Primer bases - determines whether primers are displayed simply as arrows or if you will see the bases that make up the primer on the sequence map
• Cut sites - determines if the various cut sites within the sequence are displayed on the sequence map
• Complement - determines if the sequence is shown as a single or double strand within the sequence map
• Axis - determines whether the sequence length ruler is shown on the sequence map
• Translations - determines whether translations linked to the sequence will be displayed on the sequence map
• Amino acid indices - determines whether the amino acid numbers are displayed on the sequence map (only available for AA sequences)
• Show cuts -  determines if the restriction enzymes cuts are displayed on the sequence map

Change the topology of DNA sequences

To change the topology of a plasmid from circular to linear:

1. Click the Information button on the Molecular Biology toolbar
2. Then, change Topology from Circular to Linear and click Update Information

Edit DNA and RNA sequences

Benchling's "edit sequences" functionality enables you to manually modify DNA or RNA sequences similar to a word processor, allowing for insertion and deletion of bases. This feature is valuable as it provides flexibility and direct control over sequence data, making it easy to make precise adjustments as needed during experimental design or analysis. 

It targets workflows such as cloning, primer design, and any other process requiring direct manipulation of DNA RNA sequences. 

1. Navigate to the sequence you would like to edit and select the Sequence map sub-tab
2. To manually insert bases, click on the position within your sequence where you would like to insert
3. Type or paste (cmd+V or ctrl+V) in the bases you wish to add, and click ENTER in the resulting modal
   • You can also copy and paste regions of a sequence and paste them into other regions of the same or another sequence and this will preserve annotations
4. To delete bases, highlight the region of your sequence you would like to delete by clicking and dragging your mouse
5. You can also highlight an annotation by clicking on it. Next, either press the delete button on your keyboard, or right-click and select Delete Bases

Update a linear DNA sequence start index

Indexes on DNA sequences help assess the relative location of bases on your linear and circular sequences. Keep your indexes updated in Benchling to more easily manage your sequences. 

When importing linear sequences from databases, Benchling maintains the genomic coordinates of the sequence. For sequence files uploaded using a different method, all sequence indexes begin at 1.

To update a linear sequence to match genomic coordinates:

1. When viewing a DNA sequence, click the Information button at the bottom of the right-side menu to open the Information panel
2. Enter the new start coordinate in the Start Index field
3. Click Update Information
   • The updated genomic coordinates are reflected in the numbering on the sequence axis. The first number is re-labeled as the new Start Index number

Update the start index of a circular sequence

Reindexing of circular sequences works similarly but is accessed differently. It can be done from the sequence map, linear map, or plasmid views:

1. Right-click the base to re-index and select Re-index
2. In the modal, enter the axial location you want the location to inherit. Then click Re-index
3. Confirm your selected location in the Current Location field
   • For example, if you enter 75 into the New Location field, the selected current location becomes the 75th base in the sequence, and the location of all other bases adjusts accordingly.

Export DNA and RNA sequences (.gb, .fasta, .csv, .svg, .rdf, .zip)

Benchling's export sequences functionality enables you to export individual or multiple DNA or RNA sequences in various common file formats. This feature is valuable because it allows you to share your sequence data with collaborators, transfer it to other software, or archive it for future use, ensuring data portability and interoperability. 

Export an individual sequence

To perform a single sequence export: 

1. Click into your object and click on the Information icon located on the Molecular Biology Panel
2. Scroll to the bottom of the Sequence Info modal to set export format and choose parameters
3. Click Download to export your sequence 

Note: This is the only interface where you can export your sequence as Vector Linear Map (.svg), SINGLE FASTA (.fasta), or SBOL RDF (.rdf) formats.

Export multiple files from expanded view

Benchling supports easy bulk file export in the expanded view from Global search:

1. Click the Global Search icon
2. On the side tab that pops up click the Arrow Icon to pull up the expanded view
3. Use the checkboxes on the leftmost side to bulk-select only RNA sequences
4. Once your files are selected, click on the export icon in the top right corner
5. From here you can either export your files as Multipart Genbank (.gb), Multi-FASTA (.fasta), or CSV (.csv), or as individual genbank (.gb) files in a ZIP folder

Export sequences in bulk

To export your sequences in bulk, use the Export Data modal. 

Note: Certain enterprise tenants do not have the Export Data modal enabled due to company policy.

1. To access this modal, click on your avatar (located on the bottom-left corner of the navigation bar), and select Data Export
2. In the Export Data modal, click on the Sequences export option. In this pop-up, you can designate the locations from which you'd like to export your sequences and also whether or not you'd like to include archived sequences (by reason) in your export
3. Finally, you can designate whether or not you'd like your files as individual Genbank (.gb) files in a ZIP folder or as a single multipart Genbank (.gb) file

Share a sequence file

Once a sequence has been imported:

1. Click on the sequence file that you’d like to share, click the Share button, and copy the Read-only access link
2. Send the link to the person you wish to share this sequence with
3. Anyone with the link will now be able to view your sequence (provided they have an account on the tenant), but will not be able to edit or analyze the sequence
4. You can also turn off link sharing by clicking the Turn off link sharing button

Lock Sequence(s)

Individual sequences can be locked to prevent accidental edits by you or your collaborators. The bases, annotations, primers, and translations of a locked sequence cannot be edited. Linked analyses and metadata can still be updated.

To lock a sequence:

1. Select the sequence you intend to lock
2. Click on the Information icon in the Molecular Biology toolbar
3. Select Yes next to Locked and click Update Information
4. Locked sequences will display an indicator and cannot be edited until unlocked by an authorized user
5. To unlock a locked sequence open the same menu and select No next to Locked

Note: Any user who has WRITE access to a sequence can lock or unlock it.

Note: You can also lock projects. Locking a project will lock all of its current sequences and new ones you add. You can still unlock individual sequences in a locked project.

Frequently asked questions

Q: Can I add overhangs on my sequences?

A: Benchling does not currently support overhangs on sequences. However, you can specify overhangs on attached primers.

Q: Can I annotate methylation on my DNA sequences?

A: Benchling does not currently support methylation sites.