IMonitor: A Robust Pipeline for TCR and BCR Repertoire Analysis

Abstract

The advance of next generation sequencing (NGS) techniques provides an unprecedented opportunity to probe the enormous diversity of the immune repertoire by deep sequencing T-cell receptors (TCRs) and B-cell receptors (BCRs). However, an efficient and accurate analytical tool is still on demand to process the huge amount of data. We have developed a high-resolution analytical pipeline, Immune Monitor ("IMonitor") to tackle this task. This method utilizes realignment to identify V(D)J genes and alleles after common local alignment. We compare IMonitor with other published tools by simulated and public rearranged sequences, and it demonstrates its superior performance in most aspects. Together with this, a methodology is developed to correct the PCR and sequencing errors and to minimize the PCR bias among various rearranged sequences with different V and J gene families. IMonitor provides general adaptation for sequences from all receptor chains of different species and outputs useful statistics and visualizations. In the final part of this article, we demonstrate its application on minimal residual disease detection in patients with B-cell acute lymphoblastic leukemia. In summary, this package would be of widespread usage for immune repertoire analysis.KEYWORDS next generation sequencing; bioinformatics; immune repertoire; TCR/BCR T HE diversity of T-cell receptors (TCRs), B-cell receptors (BCRs), and secreting form antibodies makes up the core of the complicated immune system and serves as pivotal defensive components to protect the body against invading virus, bacteria, and other pathogens. The TCR consists of a heterodimeric ab chain (95%, TRA, TRB) or gd chain (5%), while the BCR is assembled with two heavy chains (IGH) and two light chains (IGK or IGL). Structurally, each chain can be divided into the variable domain and the constant domain (Lefranc and Lefranc 2001a,b). The diversity of the TCR and BCR repertoire is enormous, owing to the process of V(D)J gene rearrangement, random deletion of germline nucleotides, and insertion of uncertain length of nontemplate nucleotides between V-D and D-J junctions (TRB, IGH) or V-J junctions (TRA, IGK, IGL). In humans, it has been estimated theoretically that the diversity of TCR-ab receptors exceeds 10 18 in the thymus, and the diversity of the B-cell repertoire is even larger, considering the somatic hypermutation (Janeway 2005;Benichou et al. 2012). The T-and B-cell repertoire could undergo dynamic changes under different phenotypic status. Recently, deep sequencing enabled by different platforms including Roche 454 and Illumina Hiseq (Freeman et al. 2009;Robins et al. 2009;Wang et al. 2010;Fischer 2011;Venturi et al. 2011) has been applied to unravel the dynamics of the TCR and BCR repertoire and extended to various translational applications such as vaccination, cancer, and autoimmune diseases.Several tools and software have been developed for TCR and BCR sequence analysis, including iHMMune-align (Gaeta et al. 2007), HighV-QEUST (Li et al. 2013), IgBLA...

Clonal Characteristics of Circulating B Lymphocyte Repertoire in Primary Biliary Cholangitis

“…Among the 27 noncirrhotics, 10 were recently diagnosed before ursodeoxycholic acid (UDCA) treatment, and 17 were previously diagnosed with PBC and had been treated with UDCA for .12 mo (13-15 mg/kg/d). Among the 16 cirrhotic PBC patients, 13 were UDCA treated and 3 UDCA untreated (Table I). …”

“…Primers (12 targeting V and 5 targeting C gene segments), with a minimal and non-self-hybridizing primer set, were devised to acquire maximum coverage of a heterogeneous set of target sequences of V and C families, and amplification bias was evaluated and reduced to a minimum (13). Details of primers were filed as a Chinese patent (CN103184216A).…”

IMPre: An Accurate and Efficient Software for Prediction of T- and B-Cell Receptor Germline Genes and Alleles from Rearranged Repertoire Data

“…First, the probability of V(D)J insertion/deletion at a gene terminus in an individual tends to decrease with increasing length of the deletion (14, 15). We analyzed our data (Table S1 in Supplementary Material) on the distribution of length of the V/J deletion (Figures S1A,B in Supplementary Material).…”

“…The method to generate simulated BCR repertoire sequences were described in the previous paper (14). Here, we create 12 datasets and each one includes 10 5 rearranged sequences.…”

“…TRB samples were sequenced using HiSeq 2000 with PE 150 bp kits (Table S1 in Supplementary Material). All sequence preprocessing was done and PE reads merged using IMonitor (14). …”