Ig-seq: Deep sequencing of the variable region of Atlantic salmon IgM heavy chain transcripts

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Abstract

Immunoglobulin M plays a key role in systemic protection of Atlantic salmon against pathogens. Until recent, studies have focused on antigen-specific antibodies and little is known about the IgM repertoire: its size, developmental changes and responses to antigens. We report the development of deep sequencing protocol to characterize the repertoire of IgM heavy chain variable region. Its structure and changes were examined at the early stages of life and after infection with virus of cardiac myopathy. Clonotypes are identified by the V and J gene segments and amino acid sequences of CDR3, which determine the contribution of the heavy chain to the antigen binding properties. A major fraction of transcripts are functional while the rest are either sterile (transcribed from noncoding parts of Ig loci) or include stop codons. Despite marked difference in frequencies of combinations of V and J genes, the size of repertoire is large. The IgM diversity steadily increases after hatch followed with temporal reduction during smoltification and recovery after seawater transfer. Most clonotypes are present only in one fish. However multiple transcripts in uninfected fish are produced exclusively from a small fraction of shared clonotypes. While only 4.7% of clonotypes are detected in three and more fish, they comprise 35% of transcripts. Increased frequencies of most abundant clonotypes were detected in the head kidney and blood at ten weeks after viral infection and all were shared. Occurrence of the same clonotypes in multiple individuals can be explained with either their simple structure or exposure to common antigens. Complexity of CDR3 assessed by contents of non complementary nucleotides is slightly lower in shared clonotypes but difference is small. High nucleotide diversity of CDR3 with identical amino acid sequences suggests selection.

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