.. _Primers: Isotype and Primer Annotations ================================================================================ Assigning isotype annotations from the constant region sequence -------------------------------------------------------------------------------- :ref:`MaskPrimers` is usually used to remove primer regions and annotate sequences with primer identifiers. However, it can be used for any other case where you need to align a set of short sequences against the reads. One example of an alternate use is where you either do not know the C-region primer sequences or do not trust the primer region to provide an accurate isotype assignment. If you build a FASTA file containing the reverse-complement of short sequences from the front of CH-1, then you can annotate the reads with these sequence in the same way you would C-region specific primers:: MaskPrimers.py align -s reads.fastq -p IGHC.fasta --maxlen 100 --maxerror 0.3 \ --mode cut --revpr --pf C_CALL where :option:`--revpr ` tells :ref:`MaskPrimers` to reverse-complement the "primer" sequences and look for them at the end of the reads, :option:`--maxlen 100 ` restricts the search to the last 100 bp, :option:`--maxerror 0.3 ` allows for up to 30% mismatches, and :option:`-p IGHC.fasta ` specifies the file containing the CH-1 sequences. The name of the C-region will be added to the sequence headers as the ``C_CALL`` annotation, where the field name is specified by the :option:`--pf ` argument. An example CH-1 sequence file would look like: .. literalinclude:: ../workflows/data/IGHC.fasta :language: none :download:`Download IGHC.fasta <../workflows/data/IGHC.fasta>` .. seealso:: Constant region reference sequences may be downloaded from `IMGT `__ and the sequence headers can be reformated using the :program:`imgt` subcommand of :ref:`ConvertHeaders`. Note, you may need to clean-up the reference sequences a bit before running :ref:`ConvertHeaders` if you receive an error about duplicate sequence names (e.g., remove duplicate allele with different artificial splicing). To cut and reverse-complement the constant region sequences, use something like `seqmagick `__.