How to Align Peptides

Every peptide SAR Slide is built on an alignment — the position-by-position frame that lets Ideation compare peptides of different lengths and compositions. This guide covers the ways to get one — the two computed at import, the two aligners you can run yourself, and importing a file — how to follow an alignment while it runs, how to read the warnings an imported file can raise, how to repair those warnings in the alignment editor, and how to correct which branch a side chain belongs to.

To decide which alignment suits your series, see Understanding Peptide Alignment.

Where alignments live

An alignment belongs to the dataset, not to a report. One dataset can hold as many alignments as you like, and each SAR Slide picks one of them — so two slides in the same report can use different alignments over the same peptides.

You manage them from the Alignment section, which appears in three places:

• the Alignments tab of the dataset page, to create, follow, open and delete them outside any report;
• the Create New SAR Report dialog, when you are creating a report;
• the Alignment tab of the SAR Report settings (Settings → Alignment), once a report is open.

All three show the same list of alignments, newest first. Each row carries:

• a source badge — Imported for a file you uploaded, Center-star or PepSeA for an alignment Ideation computed (hover the badge for a one-line description);
• the alignment's name, and a status when it is not complete: In progress, Failed or Replaced;
• a summary line: date · N peptides · N positions (· x/y branches aligned, for datasets with branches), followed on a PepSeA row by a settings icon you can hover to see the strategy and gap penalty it ran with.

The icons on the right open the alignment editor (Edit alignment, on a completed alignment), show the progress or the failure of a computed one, take a replaced request to the alignment that replaced it, and remove the alignment (Delete alignment).

Option 1: Use the alignments created at import

Every peptide dataset arrives with two alignments queued for it: a Center-star alignment and a PepSeA alignment. They are computed in the background once the dataset is ready, so the dataset opens straight away and the two rows show In progress until they complete. The center-star one usually finishes first; the PepSeA one can take longer, as it may wait for the monomer similarity matrix to be rebuilt for the monomers the import brought.

When you create a SAR Report, the newest completed alignment is preselected, so you can generate a SAR Slide without providing any file.

Both are best-effort: an alignment that cannot run ends as a Failed row with its reason, and the import still succeeds. Center-star does not run on datasets above 5,000 peptides or with no HELM sequence; PepSeA does not run on datasets with more than 208 distinct non-natural monomers (the default limit). If neither completes, use one of the options below.

Option 2: Align with center-star

Click New Alignment and choose Align with center-star ("Alignment computed from the dataset's own sequences with the center-star algorithm.").

An Alignment queued message confirms the request, a new Center-star alignment row appears in the list, and its progress opens. Center-star asks nothing: it derives its gap costs from the dataset itself.

Use this when the dataset was imported before automatic alignment existed, when the import-time alignment failed, or when you want a fresh computed frame to compare against a curated one. For how it works, see Understanding Center-Star Alignment.

Option 3: Align with PepSeA

Click New Alignment and choose Align with PepSeA ("Alignment computed by MAFFT through PepSeA, using the tenant similarity matrix."). If the entry is greyed out, its caption says why — the dataset uses more distinct non-natural monomers than PepSeA supports.

The alignment is queued at once, and the PepSeA alignment parameters dialog opens. The similarity matrix is already being prepared while you choose, so taking your time here does not delay the result.

1. Under Alignment strategy, keep Accurate for a normal SAR series, or pick Automatic or Fast for a large dataset. Above 200 peptides, Accurate shows a warning recommending another strategy; you can still run it.
2. Under Gap penalty, keep the default Compact, move to Very compact for a tighter frame, or down to Loose for a looser one.
3. Click Start Alignment. A PepSeA alignment started message confirms it, and the alignment's progress opens.

Cancel discards the queued alignment. The What do these do? link in the dialog opens Understanding PepSeA Alignment, which explains each setting and which strategy suits which dataset.

Running PepSeA again while a previous PepSeA request on the same dataset is still queued replaces that request: its row is marked Replaced and links to the new one.

Follow an alignment in progress

A computed alignment runs in the background, so you can keep working while it does. Its row reads In progress; click the progress icon on the row to open the steps it goes through:

Aligning branches counts the branch groups as it goes, and is left out for datasets without branches. A PepSeA alignment waiting for the similarity matrix shows Queued until the matrix is ready.

The progress closes on its own when the alignment completes, and the row's Edit alignment icon appears. If it fails, the row reads Failed: click its red icon to read why, delete the row, and run a new alignment. The most common reasons are explained in Understanding PepSeA Alignment and Understanding Center-Star Alignment.

Option 4: Import an alignment from a file

Click New Alignment and choose Import from a file ("Use an alignment you already have, as CSV or Excel").

File format

The layout is the same for CSV and for each Excel sheet:

• Column 1 holds the structure's primary identifier — the ID Ideation matched your peptides on at import.
• Every following column is one alignment position. The header labels the position; they must all be non-empty and unique.
• Each cell holds one monomer symbol. A hyphen (-) is a gap; an empty cell is read as a gap too.
• A file needs at least 2 columns (the ID plus one position).

ID,1,2,3,4,5
PEP-001,A,G,V,-,L
PEP-002,-,G,V,A,L

Use the Download template: CSV | Excel links in the dialog to start from a valid file. The Excel template ships with two sheets, backbone and branch_1, to show the multi-sheet layout.

Step 1: Choose the file and name it

Pick the file and fill in Alignment Name — it is pre-filled from the filename, and it is the name you will see in the Available Alignments list, so make it meaningful.

A CSV, or an Excel file with a single readable sheet, imports from here in one click. A multi-sheet Excel workbook walks two more steps first.

Step 2 (Excel): Choose the backbone sheet

Ideation cannot guess which sheet is the backbone, so it asks: "Which sheet holds the backbone alignment?" Each candidate is listed with its shape, so you can recognise it by size.

Choosing No backbone sheet, align it to left tells Ideation to rebuild the backbone frame from the dataset's own HELM sequences and treat every sheet in the file as a branch sheet.

Sheets that could not be read at all are listed under Ignored sheets: with the reason.

Step 3 (Excel): Map the branch sheets

Ideation detects the dataset's real branches on the chosen backbone frame and asks you to say which sheet aligns each one. Every branch gets a dropdown listing the remaining sheets plus Align at left.

A sheet can align at most one branch, so assigning it to a branch releases it from any other. Sheets you leave unassigned are called out explicitly — "Sheet "branch_2" is not mapped to any branch and will not be imported." — rather than being dropped silently.

Click Import Alignment to finish.

Review the import warnings

Importing is non-blocking. A file that does not perfectly agree with the dataset is still imported: the peptides that match are stored, and every discrepancy is recorded as a warning on the alignment. Only genuinely unusable input fails — a file that cannot be parsed, or one where no identifier matches any structure in the dataset.

So the import ends one of two ways:

• Alignment imported successfully — nothing to review.
• Alignment imported with warnings — review them in the alignment list — the alignment is there, and its row now carries an amber warning icon.

Click the amber icon (View import warnings) on the row to reopen the Import Warnings dialog at any time — the warnings are stored with the alignment, not just shown once.

Only the first 10 identifiers are listed in each message, followed by "(and N more)".

Fix a sequence mismatch in the alignment editor

A sequence mismatch is the one warning you repair directly in Ideation, and the alignment editor is built for it. Open it with the pencil icon (Edit alignment) on the alignment's row.

The table takes the left of the editor. A side panel on the right holds three collapsible sections: Sequence mismatches (shown only while some peptides matching the Filters differ from their dataset sequence), Configuration and Filters. The sliders icon at the right end of the toolbar hides or shows the panel, and dragging its left edge makes it wider or narrower.

The editor makes each disagreement visible:

• Affected rows carry an amber warning icon ("This alignment differs from the dataset sequence").
• The offending cells have a coloured border, one colour per kind of mismatch: orange for a replacement, blue for an addition, and red for a deletion (on the cell the missing monomer belongs before). A cell that is both a replacement and the cell before a missing monomer shows the replacement colour, and a monomer missing from the end of a sequence has no bordered cell. The per-kind Fix buttons use the same colours, so they also serve as the legend.
• The Sequence mismatches section, at the top of the side panel on the right, has a Show list to display only the peptides with warnings, or only those with replacements, additions or deletions. The kind options and Only the N peptides with warnings show their peptide count; All peptides has none. A kind with no peptides left is greyed out. If the kind you selected runs out of peptides, for example after you fixed them, the table shows all peptides again; an undo that brings the kind back restores your choice.
• Its Highlight mismatches switch removes the amino acid colours from the table, so the coloured borders are the only colour left.

Repair everything at once

In the Sequence mismatches section, click the Fix All button, which reads the count, for example Fix All 3 peptides with warnings (or Fix All 1 peptide with a warning). Ideation applies every repair needed to make the shown rows match their dataset sequences: wrong monomers are replaced, surplus ones removed, and absent ones inserted. It reports what it did — "Fixed N rows. Save alignment to persist." — and the repairs are yours to review before you commit them.

Repair only some kinds of mismatch

Each difference between the alignment and the dataset sequence is one of three kinds, named from the alignment's side:

Below Fix All, one button per kind (Fix Replacements, Fix Additions, Fix Deletions) repairs only that kind, and shows how many shown peptides need it. Hover a button to see what the kind means.

The kinds follow how the alignment lines up with the dataset sequence: a run of different monomers of the same length counts as replacements, not as additions and deletions. After a partial fix, the remaining differences are counted again, so their kinds can change.

Edited rows stay on screen

Editing a peptide never hides it, even when the edit makes it stop matching the Show list or the Filters: the row stays, faded, so you can check the result of your edit. An icon next to its name says what it no longer matches: a crossed-out filter for the Filters, a crossed-out eye for the Show list.

Fixes, gaps and undo all work on faded rows, and an undo or a later edit that makes the row match again brings back its colours. The bulk actions still act on every row on display, faded ones included.

A position filter stays on its position number: adding or removing gaps moves the monomers, not the filters. An edit that removes a filtered position altogether, for example by removing the gaps of the last position, removes its filter too, and the table is filtered again.

The table is filtered again only when you change a filter or the Show list, switch to another chain (the backbone or a branch), or click Re-apply filters at the top of the Filters section. Re-applying hides the faded rows and shows the peptides that match now. The button is enabled while re-applying would change the table for the Filters: a row is faded for the Filters, or a hidden peptide matches again, which an undo or redo can do. A row faded for the Show list alone does not enable it; it goes the next time the table is filtered again, for example when you change the Show list.

Repair one cell at a time

Click (or right-click) any cell to open its menu. Repair entries appear only when they apply to that cell:

Read the branch markers

Two red markers tell you where a side chain joins the backbone, one on each side of the bond:

The asterisk's corner tells you which end of the chain is bonded: top-left when the branch joins through its first residue, top-right when it joins through its last. Leading gaps do not count — a branch padded with a gap still marks its first real residue on the left. Both markers, and the bridge and chemical-object indicators alongside them, follow your gap edits as you make them.

Correct which branch a side chain belongs to

Ideation groups side chains into branches by where they attach and what they look like, and that grouping is a guess: two peptides whose side chains you consider the same series can land in different branches, and one branch can hold chains you would rather separate. You correct that here rather than by re-importing.

Click (or right-click) the backbone cell a branch hangs off — the cell marked with the branch badge — and the menu offers Reassign branch. (When a cell carries more than one branch, each gets its own entry, named after it: Reassign Branch 2.) Expanding it lists where the side chain can go:

Only moves that change something are listed, so the action never appears to do nothing: a branch the peptide already carries is not offered (its side chain is already there), and New branch appears only when the branch being left has other members — moving its last member would simply rename the branch. A branch with no move left to offer shows no entry at all.

The change is local until you save, like every other edit in the editor.

Right-clicking a position header instead gives you column-wide actions: Add gap before this position for all peptides (the clicked column and everything after it move one position to the right), Remove this position gaps for all peptides (for currently displayed peptides when a filter is active), sorting, Remove filter, and Set as first position.

Customize position numbering

The Position numbering settings, in the Configuration section of the side panel, let you:

• Set the first position: Choose which column displays as position 1 (or 0); you can also right-click a position header and pick Set as first position. It is only available for the backbone: a branch is always numbered from its own first monomer, and on a branch tab the setting reads Only available for backbone.
• Start numbering at 0: Toggle this option to show positions as 0, 1, 2, … instead of 1, 2, 3, … It applies to the backbone and to the branches (B0, B1, …), in the editor, the peptide viewer, the peptide list and the filters.

Start numbering at 0 has a default that depends on where the alignment comes from:

• A new automatic alignment starts at 0. Alignments created before this release keep numbering from 1 until you change the setting.
• An imported file starts at 0 only when its first position column is headed exactly 0, and at 1 otherwise. A file whose positions run from a negative number (for example -2, -1, 0, 1) is read as starting at 1; this is a known limitation.

These settings apply when you save the alignment, so both you and your teammates see the same numbering next time you open it.

Every change is local until you save, and the outlines disappear as soon as a cell agrees with the dataset. Use Undo (Ctrl+Z), Redo (Ctrl+Y) and Revert all changes freely.

Save

• Save alignment overwrites the alignment you opened. An alignment belongs to the dataset, so this changes every SAR Report that uses it, not only yours: Ideation asks you to confirm first, and Ctrl+S opens the same confirmation as the button.
• Save as new keeps the original and stores your edits under a new name (pre-filled as "«name» - copy").

When you save, Ideation re-checks the sequences and drops the sequence-mismatch warning once every peptide agrees — that is how the amber icon clears. The other warnings describe the file you imported rather than the sequences, so they stay on the alignment. Re-checking also happens on Save as new, so copying an alignment cannot be used to shed a warning it still deserves.

Next steps

• Understanding Peptide Alignment — the three sources compared, and which one to use
• Understanding Center-Star Alignment and Understanding PepSeA Alignment — the methods behind the computed alignments
• How to Create a Peptide SAR Report
• How to Explore the Peptide Viewer
• Understanding Peptides in Ideation