In recent years, digitalization and computer-based technologies have greatly revolutionized the field of bioinformatics. Advance research and development of computer-based programs have enhanced various DNA sequencing technologies. This advancement has significantly broadened our understanding of genomic evolution and has widely contributed to the application of clinical genomics. Cancer has been one of the major causes of death across the world. Cancer is mainly caused due to the damage or changes in DNA that affect the function of genes which contain a set of instructions that control various functions of cells. This damage in genes that maintain DNA repair mechanism may lead towards genome instability allowing rapid growth of cancer.   Microsatellite instability (MSI) is one such condition characterized due to genomic alteration leading towards the failure of DNA repair mechanism in cancerous cells. MSI is found in various types of cancer but is most often found in colorectal cancer, gastric cancer, and endometrial cancer. Hence, detection of this MSI can greatly contribute towards cancer therapies and enable to plan for the best treatment. This study mainly focuses on evaluating the performance of MSI calling algorithms using targeted sequencing methods.   The literature provides a detailed outline of various topics related to MSI detection. Moreover, different computational methods like MSIsensor, MSIsensor-ct, MSIsensor-pro, MSings, MiMSI, and MSIsensor2 were used in this study for the detection of MSI in selected samples are thoroughly discussed in the methodology section. Finally, the findings of this study conclude that the MSI calling algorithms mentioned above provide accurate detection of MSI in the chosen samples. Also, these algorithms enable us to determine the MSI status of the chosen samples more precisely

Living amidst a milieu of pathogenic organisms, vertebrates are in constant threat of contracting one or the other disease. As a mechanism of protection against such ‘invasions’, the vertebrate immune system has evolved to serve two main functions. One, to generate a specific immune response against the invading pathogen (in the from of specific antibodies and cell mediated immune responses). And two, to ‘remember’ the pathogen after the first exposure and mount a heightened and quicker immune response upon subsequent encounters. This phenomenon is called immunological memory, or anamnestic response and is achieved by the generation of memory B and T cells. The generation of specific Immunological memory is indeed the most important requirement/purpose of prophylactic vaccination Though different mechanisms are known to operate to maintain memory B and T cells, some aspects are still debatable. The ‘relay hypothesis’ (Nayak etal., Immunology.102(4)(2001); Nayak R etal., Microbes. Infect.(2005)), addresses some of those key issues. It describes that antigen specific memory B cells can be maintained by the interaction of membrane bound idiotypic (Id, Ab1) and anti-idiotypic (α-Id, Ab2) antibodies on B cells. Anti-Ids binding to idiotopes on Abs (Ab1) are known to be potential regulators of immunity in a variety of diseases, such as autoimmunity, cancer as well as viral, bacterial or parasitic infections. The relay hypothesis outlines the mechanism of persistence of memory cells in the absence of persisting antigen. This is achieved through the ‘internal image’ of the antigen on the Ab2 variable region, which serves as surrogate antigen thus helping in maintenance of immunological memory even in the absence of persisting antigen. It also explains that all antigens, protein or nonprotein can be converted to the common “coinage” of internal image peptides, otherwise called peptido-mimics. Peptido-mimics that have similar binding properties to MHC as the antigenic epitope, will ensure that the antigen specific memory T cells are also maintained. Hence T cell activation could also occur in the absence of nominal antigen, a potentially important process in T-B cooperation and immune regulation. Scope and objectives of this work: To demonstrate the presence of idiotypic and cognate anti-idiotypic antibody for the given antigen  To examine the likelihood of the three dimensional structural similarity between antigen and Ab2 variable region  To demonstrate the presence of peptido-mimics of the antigenic epitope in the Ab2 variable region; and if those peptido-mimics have structural and functional similarity with antigenic peptides when bound to MHC-I To examine the (immunological) memory associated phenotype of thediotypic anti-idiotypic B cells. The antigen of choice for the current study is Heamagglutinin-Neuraminidase (HN) protein of peste des petits ruminants virus (PPRV). Idiotypic (Id, Ab1), antiidiotypic (α-Id, Ab2) hybridoma against a deletion mutant of PPRV HN were generated and characterized. These hybridoma served as surrogate B cells for the study of Id α-Id B cell interactions. Anti-anti-idiotypic (Ab3) lymphocytes were also generated by immunizing syngenic BALB/c mice with Ab2 hybridoma. Results not only indicated the interplay of idiotypic and anti-idiotypic B and T cells in this cascade but also the mimicry of the antigen by Ab2. Ab2 Mab could recognize idiotopes of anti-PPRV HN Ab1 raised in diff species of animals, thus demonstrating that Ab2 was indeed an antigen mimic that interacts with Ab1 paratope irrespective of which species the Ab1 originates from. Ab2 Mab also mimicked the antigen (Hemagglutinin-neuraminidase) in functional assays by bringing about hemagglutination. Similarly, Polyclonal Ab3 which reacts with Ab2 Mab and with antigen, inhibits hemagglutination, just as Ab1 does, albeit to a lesser extent. This suggests Ab3 has functional similarity with Ab1. It is imperative that T cells be involved in this network of B cells for the maintenance of antigen specific immunological memory. This is because B cells require T cell help in the form of cytokines for proliferation and Cytotoxic T Lymphocytes (CTLs) are needed to control the specific population of Id and anti-Id B cells to maintain homeostasis. The Ab2 hybridoma as well as soluble Ab2 stimulated the proliferation of antigen specific T cells. Similarly, Ab3 splenocytes were stimulated to proliferate by the Ab2 as well as the antigen. Peptides generated from monoclonal Ab2 heavy and light chain variable regions (VH and VL) showed structural and functional similarity to the antigenic peptides in terms of p-MHC binding. These peptides stimulated the proliferation of antigen and Ab2 specific T cells, and also triggered 4-5 times higher CTL targeted cell lysis of peptide pulsed RMAS-Kd cells, as compared to a control peptide. VH, VL and antigenic peptides stabilized MHC-I on the cell surface of the TAP deficient, RMAS-Kd cell line for upto six hrs as compared to the ‘empty’ MHC-I, which remained on the surface only for one hr. The presence of peptido-mimics in the Ab2 variable region, which have structural similarity with antigenic peptide (when bound to MHC I), was also established using insilico software tools. Antigenic peptides and VH and VL peptides were modeled onto MHC-I crystal structures using the molecular modeling software InsightII and the minimization program, CNS. Putative MHC-I binding peptides from these sequences were generated using the p-MHC-I binding prediction algorithm, BIMAS. By replacing these peptides in the respective crystal structure of MHC I and superimposing the two structures, we have tried to establish that through structural similarity in binding to MHC-I, peptidomimics have a role in the maintenance of antigen-specific CTL memory. Consequently CTL memory specific to antigenic epitope can be preserved even in the absence of antigen by its peptidomimic. Following long-term immunizations, as expected of a secondary immune response, the serum Ab1 titre was found to be higher than the titer during primary response. It was also noted that though the number of Ab1 and Ab2 cell number was comparable in the total splenocyte population, Ab1 titre in the serum was higher than Ab2, immaterial of Ag/Ab2 booster. The same trend was noticed in prolifertion assay and CTL assays when the splenocytes were stimulated by Ag/Ab2 pulsed bone-marrow derived dendritic cells (BMDCs) as APCs. That is, irrespective of immunization and boost with Ag/Ab2, Ag pulsed BMDCs stimulated the proliferation and CTL lysis of long term immunized splenocytes to a greater extent than Ab2 pulsed BMDCs. Memory markers present on B and T cell surface might help maintain their close interactions in the idiotypic network. CD27/CD70 (CD27L) might play a role in maintaining these cells in a memory state. The Id α-Id B cells in addition to being triggered through the membrane bound Id, α-Id antibodies, can also be activated through CD27/CD70 to differentiate into plasma cells upon activation by antigen. Id and α-Id B cells were demonstrated to possess the CD27 memory marker on their surface in addition to the membrane bound IgM. Antigen specific IgM and CD27 double positive cells were detected in the range of 1-3% in the total splenocyte population. In conclusion: PPRV HN immunization triggered the generation of Ab1, Ab2, Ab3 (Id, α-Id, α-α-Id) cascade through the interaction of membrane bound immunoglobulin of the corresponding B cells. Ab2 was demonstrated to be a significant structural and functional mimic of the antigen. Peptidomimics of the antigenic epitope, present in the Ab2 variable region, can serve the purpose of maintaining antigen specific T cell memory response. These findings re-confirm the importance of anti-id antibodies in the regulation of immune responses. Ever since the concept of antigen mimicry by anti-Id antibody has been confirmed by several laboratories, the utility of anti-Ids as surrogate antigens for the purpose of prophylactic vaccination has received great attention. The results of the current work are especially significant for the purpose of development of vaccines for diseases related to antigens that are very cumbersome to purify (for ex., in case of several cancers) or when it is too dangerous to immunize with the antigen itself (for ex., in case of some pathogenic organisms). The results also signify that immaterial of the nature of the antigen, their respective petidomimics can establish and maintain immunological memory.