IG and TR single chain fragment variable (scFv) sequence analysis: a new advanced functionality of IMGT/V-QUEST and IMGT/HighV-QUEST
Résumé
Background : IMGT®, the international ImMunoGeneTics information system® (http://www.imgt.org), was created in1989 in Montpellier, France (CNRS and Montpellier University) to manage the huge and complex diversity of theantigen receptors, and is at the origin of immunoinformatics, a science at the interface between immunogeneticsand bioinformatics. Immunoglobulins (IG) or antibodies and T cell receptors (TR) are managed and described in theIMGT® databases and tools at the level of receptor, chain and domain. The analysis of the IG and TR variable (V)domain rearranged nucleotide sequences is performed by IMGT/V-QUEST (online since 1997, 50 sequences perbatch) and, for next generation sequencing (NGS), by IMGT/HighV-QUEST, the high throughput version ofIMGT/V-QUEST (portal begun in 2010, 500,000 sequences per batch). In vitro combinatorial libraries ofengineered antibody single chain Fragment variable (scFv) which mimic the in vivo natural diversity of theimmune adaptive responses are extensively screened for the discovery of novel antigen binding specificities.However the analysis of NGS full length scFv (~850 bp) represents a challenge as they contain two Vdomains connected by a linker and there is no tool for the analysis of two V domains in a single chain. Methods : The functionality "Analyis of single chain Fragment variable (scFv)" has been implemented in IMGT/V-QUESTand, for NGS, in IMGT/HighV-QUEST for the analysis of the two V domains of IG and TR scFv. It proceeds in five steps:search for a first closest V-REGION, full characterization of the first V-(D)-J-REGION, then search for a second V-REGIONand full characterization of the second V-(D)-J-REGION, and finally linker delimitation. Results : For each sequence or NGS read, positions of the 5′V-DOMAIN, linker and 3′V-DOMAIN in the scFv are providedin the‘V-orientated’sense. Each V-DOMAIN is fully characterized (gene identification, sequence description, junctionanalysis, characterization of mutations and amino changes). The functionality is generic and can analyse any IG or TRsingle chain nucleotide sequence containing two V domains, provided that the corresponding species IMGT referencedirectory is available. Conclusion : The“Analysis of single chain Fragment variable (scFv)”implemented in IMGT/V-QUEST and, for NGS, inIMGT/HighV-QUEST provides the identification and full characterization of the two V domains of full-length scFv(~850 bp) nucleotide sequences from combinatorial libraries. The analysis can also be performed on concatenatedpaired chains of expressed antigen receptor IG or TR repertoires.
Origine : Fichiers éditeurs autorisés sur une archive ouverte
