How to create alignments

Molly
Molly
  • Updated

Aligning two or more sequences is a bioinformatic technique used to compare sequences base-by-base or amino acid-by-amino acid. In Benchling, you can create alignments and save them to sequences for future reference. Consensus, pairwise, and multi-sequence alignments can be performed on DNA, RNA, and amino acid sequences. 

 

DNA/RNA alignments

Create new DNA/RNA alignment

To perform a DNA/ RNA alignment, follow these steps:

DNA Alignment GIF - Crop.gif
  1. Open the DNA/RNA alignment modal
    • Via global create: Click the Create icon in the left hand navigation bar and hover over DNA/RNA sequence, then click New DNA/RNA alignment to open the alignment modal
    • From an existing sequence: Click the Alignments button (four horizontal lines), then click Create new alignment to open the alignment modal
  2. Add sequences to be aligned using any of the methods below
    • Upload sequence or trace files
      • Accepted file types: .ab1, .ftv, .fasta, .gb, and .geneious
      • Note: RNA uploads are not supported
      • Note: For organizational purposes, we recommend aligning .ab1 files directly on the plasmid that was sequenced and viewing them there
    • Search for existing DNA/RNA sequences
    • Manually add sequence:
      • Select the nucleotide type: DNA or RNA
      • Enter a name
      • Enter the sequence
      • Click Add
  3. Click Next
  4. Choose your Alignment type
    • Pairwise alignment: Make one alignment against the template for each non-template sequence
    • Multi-sequence alignment: The results will be attached as a single alignment on the template sequence
    • Consensus alignment: A new sequence will be created with the consensus of all the selected sequences
  5. For pairwise or multi-sequence alignments, define your template sequence(s) and your non-template sequence(s)
    • Note: Multiple alignments with different template sequence(s) can be performed at once by clicking the blue plus above the template sequences
  6. For consensus alignments, define your groups of sequences
  7. Select your alignment program
    • Use MAFFT for DNA/RNA alignments
    • MAFFT-based alignment programs and parameters that can optionally be customized:
      • Auto (default): Pick a strategy automatically based on the count and size of inputs
        • Gap open penalty
        • Gap extension penalty
        • Adjust direction
      • 6-mer distance: Use 6-mer distance for constructing the guide tree
        • Mac refinement iterations
        • Tree rebuilding
        • Gap open penalty
        • Gap extension penalty
        • Adjust direction
      • Local pairwise: Compute local pairwise alignments using Smith-Waterman for constructing the guide tree and iterative refinement
        • Max refinement iterations
        • Gap open penalty
        • Gap extension penalty
        • Adjust direction
      • Global pairwise: Computed global pairwise alignments using Needlemann-Wunsch for constructing the guide tree and iterative refinement
        • Max refinement iterations
        • Gap open penalty
        • Gap extension penalty
        • Adjust direction

Note: Your selected alignment program will be saved as a preference and auto-set the next time you enter the alignment workflow

  1. Click Create Alignment

 

About plasmid alignments

Benchling allows you to use a circular DNA sequence as the template sequence to perform whole plasmid alignment or cross-origin short read alignment. Alignment sequences are automatically rotated and reindexed in the modal without affecting the sequence stored in Benchling.

If non-template sequences are all approximately (+/- 10%) the same length as the template sequence, the alignment tool assumes they are circular and attempts to pre-process the non-template sequences by optimally rotating them using the MARS tool. When the rotation is successful, the template remains linearized at the same position and all non-template sequences are rotated to match.

Note: If the template sequence is circular, reads will be treated as circular even if they are imported with an undefined topology or exist in Benchling with linear topology.

When non-template sequences are longer than the template sequence by more than 10%, the template sequence is doubled within the alignment so no reads cross the origin. If there is complete coverage of the template sequence, the alignment will remain linearized and split any cross-origin rows so the middle portion is gaps.

 

View previously performed alignments

If you created your alignment from sequences within Benchling, open one of those sequences and click the Alignments icon in the Molecular Biology toolbar on the right side of the screen. Under the Saved alignments section, click the alignment you would like to open.

If you created the alignment by uploading DNA/RNA sequence files, navigate to the folder you saved the alignment to and click on it.

View Plasmid Alignment Crop.gif

 

Use alignment tools

In an open alignment, you can access a variety of tools to help you analyze your aligned sequences. The list below tells you how to:

  • Find mismatches – click the left and right arrows in the alignment toolbar
  • Sort your alignment – click the Sort by dropdown and selecting your sorting method
  • Search an alignment – click the magnifying glass in the alignment toolbar or use the command+F keyboard shortcut
  • To rename a sequence row within an alignment – click Edit next to the name of the alignment, type the new Name into the textbox, then click Save
  • Increase/decrease your viewing window – drag the edge of the viewing window in/out
  • Trim an alignment – highlight the portion of the sequence you want, right click, hover over Trim…, then select how to trim your sequence. You can also drag the black bars within the sequence map to manually trim a sequence
Trim DNA Alignment GIF - ezgif.gif
  • Trimming options: to start, to end, to selection, to start (all sequences), to end (all sequences), or to selection (all sequences)
  • Note: The trimming black bars will default to before the first base and after the last base if the sequence has yet to be trimmed
  • Note: The trimmed sections will be grayed out and are ignored when you realign
  • Realign the sequences to do any of the following by clicking Realign in the upper right:
    • Update a sequence after editing it within Benchling
    • Update your alignment to remove the trimmed portions of your sequence
    • Add new sequences to the alignment
    • Remove sequences from the alignment
    • Change the alignment program or parameters

Leave comments

Comments allow you to make notes throughout your alignment. They can be viewed and edited by anyone with the appropriate permissions. To add a comment to your alignment, highlight a base or region, click Add Comment from the toolbar, type your comment into the pop-up in the lower left corner, then click Save.

To remove comments:

  • Open the comment and click on the trash icon OR
  • Right click on the comment and select Delete Comment

Note: Comments are not searchable within Benchling

Adjust visibility settings

By clicking the Gear in the upper toolbar, you can toggle on/off the following alignment visibility settings to assist you in analyzing the alignment:

  • Annotations
  • Translations
    • Click the Gear to change the amino acid coloring
  • Amino acid indices
  • Reading frames
    • Click the Gear to change which frames you are viewing
  • Primers
  • Alignment axis
  • Sequence axis
  • DNA
  • Trace
  • Trace quality
  • Quality-based capitalization
  • Votes
  • Row statistics
  • Comments
  • Expanded mini-map
  • Expanded view

By clicking the Down carat to the left of each sequence name, you can toggle on/off the following visibility settings on individual sequences to assist you in analyzing the alignment:

  • Annotations
  • Translations
    • Click the Gear to change the amino acid coloring
  • Amino acid indices
  • Primers
  • Alignment axis
  • Sequence axis
  • Votes
  • Comments
  • Expanded mini-map
  • Expanded view

 

Export alignment

To export your alignment:

  1. Click the Export dropdown in the upper right corner
  2. Select the file type for your export:
    • Available file types:
      • Clustal
      • FASTA
      • PDF- Screen capture
      • PDF- Formatted
      • Alternatively, you can also copy a sharing link 

 

Amino acid/protein alignments

Create new AA alignment

To perform an alignment of amino acid sequences, follow these steps:

  1. Open the protein alignment modal
    • From global create: Click Global Create in the left hand navigation bar, hover over AA sequence, then click New AA alignment to open the alignment modal
    • From an existing sequence: Click the Alignments button (four horizontal lines) then click Create new alignment to open the alignment modal
  2. Add existing AA sequences to the alignment by searching for them
  3. Do one of the following:
    • Perform a template alignment by hovering over a sequence name and clicking Set Template
    • Perform a consensus alignment by ensuring no sequences are set as a template
      • Note: If you open the protein alignment modal from an existing AA sequence, that sequence is automatically selected as the template. You can remove this sequence from the template by hovering over the row containing the sequence name and clicking Unset template
  4. Click Next
  5. If you opened the protein alignment modal from global create, you will be prompted to enter an Alignment name and select a Project folder to save the alignment
  6. Click Create Alignment
    • Note: Protein alignments are read only
    • Note: Protein alignments are automatically computed using Clustal Omega
AA Alignment GIF Crop.gif

 

View previously performed alignments

Open one of the sequences used in the alignment and click the Alignments button in the Molecular Biology toolbar on the right side of the screen. Under the Saved alignments section, click the alignment you would like to open.

AA Alignment GIF Crop.gif

 

Use alignment tools

In an open alignment, you can access a variety of tools to help you analyze your aligned sequences.

Realign sequences

Realign the sequences to do any of the following by clicking Realign in the upper left:

  • Update a sequence after editing it within Benchling
  • Add new sequences to the alignment
  • Remove sequences from the alignment
  • Change the alignment program or parameters

Adjust visibility settings

By clicking the Gear in the upper toolbar, you can toggle on/off the following alignment visibility settings to assist you in analyzing the alignment:

  • Axis
  • Annotations
  • Mismatches
  • Coloring scheme
    • Click the Gear to change the coloring scheme
  • Identity
  • Summary Identity
    • Click the Gear to change the sliding window size
  • Hydrophobicity
  • pI
  • Row statistics
  • Expanded mini-map
  • Antibody numbering
    • Click the Gear to change the numbering scheme
    • Note: Antibody numbering is performed using the SAbPred/ANARCI library (Dunbar and Deane, 2016)
  • CDR annotations
    • Click the Gear to change the CDR definition
    • Note: The antibody numbering is the basis for identification of CDRs
  • Liability sites
    • Click the Gear to change which liability sites are identified
    • Liability site patterns and references (hyperlinked):
      • N-link glycosylation pattern: *N_[S|T]
        *Where the blank is any single AA except Pro
      • Asparagine deamidation pattern: NG|NS|NT|NN
      • Asparagine deamidation (in CDRs) pattern: **N
        **Instances of deamidation modifications on non-canonical motifs have been documented across all CDRs. Enabling this site will flag all Asn’s that reside within an identified CDR region, as defined by your CDR annotations visibility setting
      • Methionine Oxidation pattern: M
      • Tryptophan Oxidation pattern: W
      • Aspartate isomerization pattern: DG|DS|DT|DD|DH
  • Expanded View

By clicking the Down carrot to the left of each sequence name, you can toggle on/off the following sequence-specific visibility settings to assist you in analyzing the alignment:

  • Axis
  • Annotations
  • Identity
  • Hydrophobicity
  • pI
  • Expanded mini-map
  • Antibody numbering
    • Click the Gear to change the numbering scheme
    • Note: Antibody numbering is performed using the SAbPred/ANARCI library (Dunbar and Deane, 2016)
  • CDR annotations
    • Click the Gear to change the CDR definition
    • Note: The antibody numbering is the basis for identification of CDRs
  • Liability sites
    • N-link glycosylation pattern: *N_[S|T]
      *Where the blank is any single AA except Pro
    • Asparagine deamidation pattern: NG|NS|NT|NN
    • Asparagine deamidation (in CDRs) pattern: **N
      **Instances of deamidation modifications on non-canonical motifs have been documented across all CDRs. Enabling this site will flag all Asn’s that reside within an identified CDR region, as defined by your CDR annotations visibility setting
    • Methionine Oxidation pattern: M
    • Tryptophan Oxidation pattern: W
    • Aspartate isomerization pattern: DG|DS|DT|DD|DH
  • Expanded view

 

View alignment statistics

On the left side of the bottom navigation panel, the following information about your alignment is automatically calculated:

  • Length
  • Number of proteins
  • Identical sites
  • Pairwise identity

This information is also available for each non-template sequence row of the alignment by looking within the grey box on the left hand side.

 

Export alignment

To export your alignment:

  1. Click the export dropdown in the upper right corner of the alignment
  2. Select what file type to export your alignment to
    • Available file types:
      • FASTA
      • PNG
      • PDF
      • Alternatively, you can also copy a sharing link 

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