Mutation analysis of IGH associated with different isotypes

Immunoglobulin heavy chain rearrangements were sequences from PBMCs of 8 individuals using IGHC primers for all isotypes bar IgE and IgD. Sequencing was carried out on the 454 pyrosequencing platform and rearranged immunoglobulin heavy chain sequences were partitioned against germline IGHV, IGHD and IGHJ using iHMMune-align. The datasets for each individual was filtered to remove non-productive rearrangements (frame shift of the IGHJ or stop codons), those with greater than 45 IGHV mutations, sequences which contained indels in the V or J, and those with ambiguities. Within each data only unique sequences were reatained (100% identical). Clonally-related sequences within each subject's dataset were also identified. Following germline reversion of all non-CDR3 nucleotide positions, sequence were clustered using the vmatch package (http://www.vmatch.de/) allowing up to 3 differences. A single representative for each cluster (representing a likely clonal set) was retained within the dataset. Representative sequences were selected based on the sequence with the highest copy number, or in cases where sequences shared the highest copy number, the least mutated sequence. If a cluster spanned multiple isotypes then a representative for each isotype was kept.

本数据集所包含的免疫球蛋白重链重排序列，取自8名个体的外周血单个核细胞（Peripheral Blood Mononuclear Cell, PBMC），实验使用针对除免疫球蛋白E（IgE）和免疫球蛋白D（IgD）外所有亚型的免疫球蛋白重链（IGH）引物进行扩增。测序工作在454焦磷酸测序平台上完成，随后利用iHMMune-align工具，将重排免疫球蛋白重链序列与种系IGHV、IGHD及IGHJ基因进行比对注释。随后对每个个体的数据集开展过滤处理：移除无功能性重排序列（即存在IGHJ移码突变或终止密码子的序列）、IGHV突变数超过45个的序列、V或J区存在插入缺失变异（Insertions and Deletions, indels）的序列，以及存在序列歧义的序列。每个数据集中仅保留完全同源的唯一序列（100%一致）。同时鉴定每个受试者数据集中的克隆相关序列。在将所有非互补决定区3（Complementarity Determining Region 3, CDR3）的核苷酸位点回复为种系序列后，使用vmatch工具包（http://www.vmatch.de/）对序列进行聚类，允许最多3个碱基差异。每个聚类（代表潜在的克隆组）仅保留一条代表序列纳入最终数据集。代表序列的选择标准为：优先选取拷贝数最高的序列；若多个序列拷贝数相同，则选择突变最少的序列。若某一聚类涵盖多种免疫球蛋白亚型，则为每个亚型分别保留一条代表序列。