How to characterize sequences with annotations and translations

You can denote regions of interest (ex. Genes, ORFs, etc.) on DNA, RNA and amino acid sequences by adding annotations and editing existing ones. You can also customize their informational fields such as annotation type and display colors, and delete annotations you don’t need.

The translations list allows you to view and manage all the amino acid translations of a given DNA or RNA sequence. It provides an organized way to interact with the protein sequences encoded by your nucleotide sequence.

The feature library in Benchling is a standardized repository of common biological features, such as promoters, terminators, protein tags, and restriction sites. Instead of manually identifying and labeling features every time a new sequence is created, a feature library allows you to automatically apply these annotations. It's a key tool for improving standardization, reproducibility, efficiency, and collaboration across a research organization.

All the annotation and translation tools can be accessed from the features menu of the Molecular Biology toolbar from either the annotation or translations tab.

How sequence features are defined when importing and exporting 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

Annotations

Annotations are features marked on sequence entities. The following are core components:

Name: The descriptive name given to the annotated feature (e.g. "pBR322 ori")
Type: A classification for the annotation, which helps to categorize different features (e.g., "promoter," "terminator," or "origin of replication"). Benchling offers default types, but you can also create custom types
Location (Start/End): The precise start and end coordinates of the annotated region on the sequence. For circular plasmids, this may wrap around the entire sequence
Strand Orientation: Indicates whether the feature is on the forward or reverse strand of the sequence. This is essential for features like coding sequences (CDS) or primer binding sites
Color: A customizable color that visually represents the annotation on the sequence map, making it easy to identify and distinguish different features at a glance
Custom Fields: You can add additional, user-defined fields to an annotation to store more specific metadata. These can include things like a description, a link to a related protocol, or the source of the feature

Access a list of annotations on your sequence

When you are working with a sequence in Benchling, you can create, view, edit, and delete annotations in the Features panel and its expanded view. To access the Features panel, make sure you have a sequence open and click the flag icon in the Molecular Biology toolbar.

You can view all the annotations on a sequence in a larger window by clicking Show Expanded View in the Features panel. This opens a full list of annotations in an Annotations tab where you can more easily view and manage them.

Create annotations

To add annotations to a DNA, RNA or amino acid sequence:

Highlight the region of interest on the sequence
Open the Features panel (Flag icon) or right-click the highlighted region and select Create new at the top of the panel
Use the text box to enter a name for the annotation
You may also edit the various components of the annotation listed above
Click Save

Add custom fields to annotations

If there is other information you would like to capture on an annotation, this can be done by adding custom fields. To add custom fields:

In the Features panel, click an annotation to expand it
Click + next to Custom fields
Enter the field name to search for a Benchling-generated field or create a new custom field
Click Save

Change the color of an annotation

To change the color of an annotation:

Open the Features panel
Click an annotation to expand it
Select a color from the Color drop-down menu

Edit annotations

If you need to edit annotations in Benchling to correct errors or customize your data for specific experiments (changing names, color, modifying position), features like the feature library and auto-annotation are powerful for standardizing data, manual editing remains an essential part of the molecular biology workflow.

Open the Features panel
Click on an annotation to expand it
Edit the name, position, annotation type, color, strand orientation, or custom fields
Click Save

Edit annotations in bulk

You may want to edit a common field on multiple annotations at once. To make one change apply to all selected annotations:

Open the Features panel and click Show Expanded View
Use the check boxes to select the annotations you’d like to edit
Click the edit icon in the top-right corner
This will open a modal that will let you adjust annotation type, strand, and color in bulk
Edit the fields, then click Save

Delete annotations

You can delete annotations to clean up your sequence maps, correct errors, and ensure that your data is accurate and not cluttered with irrelevant information.

Hiding annotations is recommended when an annotation is correct but you don't need to see it for a specific task. For example, if a plasmid has many annotations, you might hide all of them except for the gene of interest and your primer binding sites to focus on a particular region. Hiding is for visual organization and temporary decluttering, not data correction.

You can hide annotations using the gear symbol on the upper right corner of the sequence to toggle on and off different features of the plasmid map.

Deletion is recommended when an annotation is a genuine error, or when the feature it represents is no longer present in the DNA sequence. Deleting is more of a final action for data cleanup. However note that deleted annotations can be recovered via version history.

You can delete annotations with either of the following two ways:

Right-click an annotation on the sequence and select Delete Annotation
Click the Features icon on the mol bio bio toolbar, click Show expanded view, selecting the annotation(s) and clicking the trash can icon in the top-right corner

Create a feature library

A feature library is a collection of annotated features that can be used to automatically annotate features across multiple sequences. You can use feature libraries to standardize annotations across sequences and share your libraries with other organizations and users.

To create a feature library:

Click the flag icon to open the Features panel
In the panel, click Edit feature libraries in the bottom-right corner
Enter your library name and description, then click Create Feature Library
- You can enter individual features manually or import features in bulk

To enter individual features to a new library:

Use the text box to enter the feature name and feature type
- Note: A feature type is a label that defines what a specific annotated region of a sequence is and helps categorize and standardize annotations on a sequence ex. gene, regulatory region, primer, etc.
Use the text box to enter the bases of your feature (Note: You can use make use of regex to perform fuzzy matches)
Select a color and classification as protein or nucleotide
Click Add Feature

You can import features in bulk via .csv or ApE library file (.txt). Note that .xlsx files will result in an error. Before importing, ensure your .csv file is formatted correctly.

To import features:

Click Import CSV or Import from APE in the top-right corner
Define which feature library to add them to from the drop-down menu
Select either nucleotide or protein as the match type for rows where match type is unspecified
Use the upload box to choose your file
Click Upload
- Tip: You can create a new library from the import modal by selecting Create new library from the feature library drop-down menu

Format .csv import files

Format the .csv file with the following column headers:

Name
Feature
Type (optional)
Color (optional) - hex color codes are accepted in the format #000000

There are 16 colors to choose from when assigning colors to annotations. Hex color codes assigned to features on an import file are mapped to the closest one of these colors:

Add existing annotations to a feature library

You may want to add an existing annotation to a feature library in Benchling to standardize and save that feature for future use across their organization. This action transforms a one-off annotation into a reusable, institutional asset.

You can add annotations that exist on your sequence to a feature library in the Features panel expanded view.

To add existing annotations:

Click the Features icon in the Molecular Biology toolbar
Click Show Expanded View
Select the annotations to add to the feature library
Click Save icon in the top-right corner this will generate pop up
Select the feature library to save the annotations to, then click Save Features

Auto-annotate sequences

After creating a feature library in Benchling, you can auto-annotate DNA, RNA, and amino acid sequences based on the information stored in feature libraries. By auto-annotating a sequence you can save time, ensure data consistency and improve collaboration across an organization.

To auto-annotate sequences:

Click on the flag icon in the Molecular Biology toolbar to open the Features panel
Click Annotations at the top of the panel
Click Auto annotate at the bottom of the panel
Select which feature library or sequence to annotate your sequences from:
- Click Search All Libraries to annotate from all your feature libraries
- Enter a specific sequence or folder to extract the annotations from
Select the annotations, then click Add Annotations to display them on your sequence

Auto-annotate sequences in bulk

You can quickly annotate many sequences in bulk using your feature libraries. To bulk auto-annotate sequences:

Open the project or folder that holds your sequences and click the directional arrow to expand the window
Select the sequences to annotate
In the More drop-down menu in the top-right corner, hover over Analyze and select Auto-Annotate
Use the dropdown to select which feature libraries to use
Click Auto-Annotate

Translations

Core Components of a Translation:

Name: The descriptive name given to the translation (e.g. "puC19 promoter translation")
Position: This defines the specific region of the parent DNA or RNA sequence from which the translation is derived. It includes the start and end coordinates on the nucleotide sequence.
Genetic Code: The specific set of rules used to translate a nucleotide triplet (codon) into an amino acid. While the Standard Genetic Code is the default, Benchling allows you to use other genetic codes, such as those for mitochondria or bacteria
Color: A customizable color that visually represents the annotation on the sequence map, making it easy to identify and distinguish different features at a glance
Strand: This indicates whether the translation is from the forward or reverse strand of the parent nucleotide sequence
Custom Fields: You can add additional, user-defined fields to an annotation to store more specific metadata. These can include things like a description, a link to a related protocol, or the source of the feature

Access a list of translations on your sequence

Translations are a special type of annotation for CDS features that include the AAs for which the annotated DNA/RNA sequence encodes] You can translate DNA or RNA sequences, create amino acid sequences, and generate biochemical properties for translations.

You can view all the translations on a sequence in a larger window by clicking Show Expanded View in the Features panel. This opens a full list of translations in a Translations tab where you can more easily view and manage them.

Create a translation

To create a translation for a DNA sequence:

Highlight your region of interest or annotation on the DNA
Right click on the region and click Create translation and select Forward or Reverse as the direction
Alternatively after highlighting the region click on Features in the Molecular Biology toolbar then click the translations tab on the top of the panel, click Create New at the top of the panel
Enter the translation name and any other components of the translation
Click Save
- Tip: Alternatively, you can select a region or an annotation on the sequence, then right-click it and select Create Translation

The translation is automatically indexed. If the translation isn't indexed or to remove the indexing, click the gear icon in the top-right corner and toggle Amino Acid indices.

Add to an existing translation

If your protein is the product of multiple separate regions/exons being translated, you can use translation groups to join multiple translations together, without translating the introns:

Highlight your region of interest or annotation on the DNA
Click on Features in the Molecular Biology toolbar then click the translations tab on the top of the panel
Click Create New at the top of the panel
Click Add to existing in the middle of the panel
Select the Forward or Reverse strand
Select the existing translation from the Add as region to drop-down menu
Click Save

Regions of a translation cannot be edited once saved. Regions of a translation can be deleted from the Features panel or from the expanded view Translations tab.

Analyze a translation

To view a translation's biochemical properties, right-click a translation and select Analyze Translation.

If a translation has a non-standard genetic code, the biochemical properties reflect the translation using the non-standard genetic code.

These biochemical properties will be analyzed:

Amino acid frequencies
Net Charge
Extinction coefficients
Instability index
Molecular weight of the resulting protein
The protein's isoelectric point (pl)
Translation position relative to the sequence

You can view a translation's biochemical properties with or without translating the first amino acid as methionine by checking or unchecking Translate start codon as methionine.

Create an amino acid sequence from a translation

Creating separate amino acid entities enables customized protein-specific viewing options such as color scheme, CDR annotations, and liability sites.

To create an amino acid sequence map from a translation on a DNA or RNA sequence:

Right-click the translation and select Create AA Sequence
Select a folder, a new name, and a schema for the AA sequence
Click Create

If the translation corresponds to a non-standard genetic code, an AA sequence identical to the translation and its non-standard genetic code will be created.

Translate DNA and RNA sequences to amino acids using alternative genetic codes

You may work with sequences that originate from or are expressed in different host organisms that don’t adhere to the standard codon table; instead, you translate DNA to amino acid following alternative, or non-standard, genetic codes defined by NCBI.

By default, Benchling uses the standard genetic code unless you set an alternative one or one is defined in an imported GenBank file. You can set genetic codes when creating new translations or editing existing translations.

To set or change an alternative genetic code on an existing translation:

Open the sequence and select the translation by clicking its name
Click the Features icon on the Molecular Biology toolbar make sure you are on the translations tab
- Alternatively, right-click the translation and select Edit Translation
Select the genetic code from the drop-down menu. You can search for genetic codes by name, number, or taxonomy keywords, which are determined by the systematic range for each genetic code as defined by NCBI
Click Save. The amino acid residues in the translation update to reflect the selected genetic code

While a DNA or RNA sequence can have many translations, each translation can have its own genetic code. This means you can model a single DNA or RNA sequence to express many AA sequences using different genetic codes.

Apply alternative genetic codes to multiple translations

To apply a genetic code to multiple translations on a sequence:

Click Features icon on the mol bio tool bar, make sure you are on the translations tab
Click Show expanded view
Select the translations, then click the Edit icon in the top-right corner
In the pop-up window, select the genetic code from the drop-down menu, then click Save

Note: A translation from an alternative genetic code will only be linked if there’s already a translation on the sequence with the same genetic code.

Create AA sequences from translations with alternative genetic codes

You can create an AA sequence that uses an alternative genetic code directly from the corresponding translation by right-clicking on the translation and selecting Create AA Sequence.

Note: Creating AA sequences from the Create menu creates an AA sequence based on the standard genetic code.

Benchling currently supports all genetic codes defined by NCBI except:

27. Karyorelict Nuclear Code
28. Condylostoma Nuclear Code
31. Blastocrithidia Nuclear Code