Reconstructing germline sequences

Example Data

We have hosted a small example data set resulting from the Roche 454 example workflow described in the pRESTO documentation. The files can be downloded from here:

Change-O Example Files

The following examples use the S43_db-pass_parse-select.tab database file provided in the example bundle, which has already undergone the IMGT/IgBLAST parsing and filtering operations.

Adding germline sequences to the database

The CreateGermlines tool is used to reconstruct the germline V(D)J sequence, from which the Ig lineage and mutations can be inferred. In addition to the alignment information parsed by MakeDb to generate the initial database, CreateGermlines also requires the set of germline sequences that were used for the alignment passed to the -r argument. In the case of V-segment germlines, the reference sequences must be IMGT-gapped. Because the D-segment call for B cell receptor alignments is often low confidence, the default germline format (-g dmask) places Ns in the N/P and D-segments of the junction region rather than using the D-segment assigned during reference alignment; this can be modified to generate a complete germline (-g full) or a V-segment only germline (-g vonly) if you wish. The command below adds the germline sequence to the GERMLINE_IMGT_D_MASK column of the output database:

CreateGermlines.py -d S43_db-pass_parse-select.tab -r IMGT_Human_IGH[VDJ].fasta -g dmask

Alternatively, if you have run the clonal assignment task prior to invoking CreateGermlines, then adding the --cloned argument is recommended, as this will generate a single germline of consensus length for each clone:

CreateGermlines.py -d S43_db-pass_parse-select_clone-pass.tab -r IMGT_Human_IGH[VDJ].fasta \
    -g dmask --cloned

Note

The germline set passed to -r must contain the complete set of germlines used by the reference alignment software (IMGT/HighV-QUEST or IgBLAST). If alleles called by the aligner are missing from the reference set, they will not be successfully processed. Additionally, the V-segment reference set must contain IMGT-gapped sequences to properly reconstruct germlines, even if the reference alignment was performed on ungapped sequences.

Note

While MakeDb provides the ihmm subcommand to parse alignment output generated by iHMMuneAlign, there is insufficient information to successfully reconstruct germline sequences for all cases using CreateGermlines.

See also

The TIgGER R package provided tools for identifing novel polymorphisms and building a personalized germline database. To use the germline corrections provided by TIgGER you would replace the V-segment germline file with the one generated by genotypeFasta (-r IGHV_genotype.fasta IMGT_Human_IGH[DJ].fasta) and specify the genotyped V-segment column (--vf V_CALL_GENOTYPED):

CreateGermlines.py -d genotyped.tab -r IGHV_genotype.fasta IMGT_Human_IGH[DJ].fasta \
    -g dmask --vf V_CALL_GENOTYPED