Comparison Of The Fundamental Functions Of Immune Repertoire Analytical Tools And Its Applications

The high-throughput immune repertoire sequencing technology enables researchers to track the characteristics and changes of the immune repertoire with unprecedented depth and breadth.It has been used in the fields of infection and vaccines,tumors,autoimmune diseases,allergies,etc.,and lots of achievements have been made.However,the large batch of similar tools for high-throughput data analy sis of immune repertoire leave immunologists no choice.Secondly,how to scientifically manage known and unknown antigen receptor sequences is one of the urgent problems in this field.In addition,individualized analysis based on different research purposes and data characteristics hinders direct parallel comparison of results among similar studies.Low-level processing performance(especially gene annotation and the identification of CDR3)may highly asscociate with the reliability of the high-level processing results.This study systematically explored and compared general features and effectiveness of fundamental functions for common immune repertoire analysis tools,and gave recommendations for tool selection according to different research purposes and data characteristics.Users can select suitable tools according to the input file formats,sequencing strategy,reference sequence customization,and the advantages and disadvantages of the fundamental functions one after another.Based on the understanding of the similarities and differences between these tools as well as the major factors(including algorithms,base errors,junctional insertions/deletions,sequencing depth)for it,two distinct tools were chosen for two different high-level analysis cases.On one hand,IgBLAST was selected to establish a universal and efficient flow for the collection,screening,annotation and management of antibody sequence data from public databases.There were 88,059 non-redundant antibody sequences obtained from seven public databases by this flow,with their key features annotated with and researched.There is an obvious preference for the usage of V and J genes in the antibody sequences.The lengths of the CDR3 and variable regions of the heavy,lambda and kappa chains decrease sequentially.The length of the heavy chain is more widely distributed and dispersed.This antibody sequence database may help to quickly obtain the target sequences or annotate new sequences,and its building flow can be extended to the construction of the TCR database.On the other hand,MiXCR was selected to define and compare the key features of the antibody repertoire under four different influenza antigen stimulation statuses by a unified analysis proccess.Both steady and various influenza antigen stimulation statues presented clonal expansion,but their patterns were not the same.The networks can sensitively reflect the status of clonal expansion and its change.Consistent with previous results,the usage distribution of V family,V gene and VJ recombinations has an obvious individual genetic tendency,and there are only slight fluctuations under different stimulation statuses.IGHV3,IGHV4 and IGHV1 families together account for most of the V family of heavy chain.The length of CDR3 at different time points of the same individual has a common basic distribution pattern,while the proportions of lengths fluctuate with the change of immune status.Large clones have no obvious gene or length preference,while the combination of large clones,public clones,clone networks and mutation rates may help to screen potential antigen reactive clones.In summary,this study may promote the development and application of high-throughput immune repertoire technology through guiding the selection of analysis tools,the construction of antibody sequence database and the unified analysis of data derived from similar researches.