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Germline Analysis

View the sequence broken into Leader, Variable (V and J), and Constant regions, with top germline hits aligned below.

This tool is helpful in the evaluation of germline origins, identifying V and J gene assignments, and assessing the alignment of leader and constant domains against reference sequences.

Accessing the Tool

Select an entry in the Project View. Go to the Analysis menu and select Germline. This will open the Germline workspace in a new tab.

Germline Analysis

Using the Tool

  • Leader Section: Displays the best matching Leader sequence found via sliding identity search against the reference database, aligned with the query sequence.

  • Variable Section: Displays the sequence broken into V and J regions with their top germline hits aligned below. When multiple species are selected, a species indicator (e.g., [H] for Human, [Rh] for Rhesus, [M] for Mouse) is shown next to the germline name.

    [!NOTE] CDR3 Junction Behavior: The backend germline assigner ignores the CDR3 junction (evaluating only IMGT positions 1 to 104) when calculating V-region identity percentages and assigning the closest germline species for the main Project View. However, the alignment tables and matches shown in the Germline workspace evaluate the full V-region sequence (IMGT positions 1 to 108), including the CDR3 junction. This difference in length coverage can occasionally cause minor discrepancies in sequence identity or break species assignment ties differently.

  • Constant Section: Displays the best matching Constant domains (e.g., CH1, Hinge, CH2, CH3 for Heavy chain, or KC/LC for Light chain) aligned with the query sequence.

  • Settings Panel: Use the sidebar on the left to filter by Species (Human, Mouse, Alpaca, Cat, Dog, Pig, Rabbit, Rat, Rhesus, Chicken), Chains (Light/Heavy), Domains (Leader/Variable/Constant), and the number of germline hits to show. A legend for species indicators is provided here.
  • Excel Export: Click the Export Excel button in the toolbar to export the current view and alignments to a spreadsheet.
  • Engineering Mutation Designs: Click a residue to enter a mutation design used by the Engineering tab.
  • Observations: Click a residue to enter an observation for that IMGT position. These are displayed in the Observations tab and on a clicked residue.
  • Indicator Dots: Dots on a residue position indicate that there is an associated engineering design or observation at that position. Engineering mutations will appear as a small eggshell square while observations will appear as a small orange circle.

Supported Reference Species

The Germline tool matches sequences against reference databases compiled from the following species:

Scientific Name Common Name (AbLead Badge)
Homo sapiens Human (H)
Macaca mulatta Rhesus (Rh)
Mus musculus Mouse (M)
Rattus norvegicus Rat (R)
Oryctolagus cuniculus Rabbit (Rb)
Vicugna pacos Alpaca (Al)
Canis lupus familiaris Dog (Dg)
Felis catus Cat (Ct)
Gallus gallus Chicken (Ck)
Sus scrofa Pig (Pg)

Allotype Detection

The Germline tool automatically detects allotypes and isotypes based on specific amino acid residues in the constant domains using the EU numbering system. The detected allotypes are displayed in the page title next to the antibody name (e.g., Germline Analysis: [Name] (G1m17,1, Km3)).

Supported Allotypes and Isotypes

Chain Class/Subclass Allotype/Isotype EU Positions & Residues Human Prevalence
Heavy IgG1 G1m17 CH1 214 = K Cau: 15-35%, As: ~100%, Afr: ~100%
G1m3 CH1 214 = R Cau: 65-85%, As: ~0%, Afr: ~0%
G1m1 CH3 356 = D, 358 = L Cau: 35-40%, As: ~100%, Afr: ~100%
G1m2 CH3 431 = G Cau: 10-15%, As: <10%, Afr: <10%
IgG2 G2m23 CH1 189 = T AND CH2 282 = M Cau: ~50%, As: ~25%, Afr: ~25%
Light Kappa Km1 KC 153 = V, 191 = L Cau: 10-15%, As: ~30%, Afr: ~30%
Km1,2 KC 153 = A, 191 = L Rare in all populations
Km3 KC 153 = A, 191 = V Cau: 85-90%, As: ~70%, Afr: ~70%
Lambda Mcg+ LC 112 = N, 114 = T Isotypic marker (IGLC1)
Kern+ LC 152 = G Isotypic marker (IGLC2/IGLC3 variant)
Oz+ LC 190 = K Isotypic marker (IGLC2 variant)

Note: IgG1 markers are combined using standard notation (e.g., G1m17 + G1m1 -> G1m17,1). Prevalence values are approximate (Cau: Caucasian, As: Asian, Afr: African) and vary by specific ethnic sub-populations.

3D Structure Visualization & Residue Selection

The Germline workspace includes an interactive PDBe Mol* 3D structure viewer positioned below the grids, allowing real-time mapping of sequence changes to the three-dimensional antibody structure.

  • Interactive Resizer: Drag the horizontal divider bar directly above the 3D window up or down to adjust the vertical height of the viewer pane.

  • Cartoon Region Coloring: The ribbon structures of the light and heavy chains are colored by region to match the centralized color schemes:

    • Variable Domain (Fv): Frameworks (dim gray/silver) and CDRs (blues, purples, cyans) are colored according to domain region standards.
    • Constant Domains: Reconstructed constant domains (CL, CH1, hinge, CH2, CH3) are automatically rendered and colored by their specific sub-domain region colors.

  • Selected Residue Highlighting:

    • Clicking column headers in the alignment grids (scheme position or linear number) highlights those columns in blue.
    • The corresponding residues on the 3D structure are instantly highlighted in CPK element coloring (with position number labels), regardless of which exclusion method is active.
    • Residue Style: Toggle the dropdown to change the rendering style of selected residues between Spacefill (default) and Ball-and-Stick.

References

  • Jefferis R, Lefranc MP. Human immunoglobulin allotypes: Possible implications for immunogenicity. mAbs. 2009 Jul-Aug;1(4):310-8. doi: 10.4161/mabs.1.4.9122.
  • Lefranc M-P, Lefranc G. Human Gm, Km, and Am allotypes and their molecular characterization: a remarkable demonstration of polymorphism. Methods Mol Biol. 2012;882:635-80. doi: 10.1007/978-1-61779-842-9_34.