BXSB_mice_CD20_BZR_repertoire, Werner et al

We investigated if a short-term B cell depletion during the pre-clinical phase of SLE in BXSB mice influences the B cell receptor repertoire. We could show that male BXSB mice treated with an anti-CD20 monclonal antibody show reduced diversity for IgG VDJ recombinations. To perform this analysis Next Generation Sequencing was performed by on the Illumina MiSeq platform using a pair of overlapping paired-end reads (2x300 bp). 20 % PhiX was added for improved performance. Paired-end reads were merged with the IgRepertoireConstructor (Safonova et al., 2015). Read Quality was ensured by FASTQC quality analysis by MultiQC (Ewels et al., 2016). Molecular amplification fingerprinting (MAF) error correction was achieved with unique molecular identifiers (UMI) as part of the cDNA synthesis and Multiplex Primers. PCR sequencing errors were corrected by pattern matching and filtering sequences containing both UMIS and correcting sequencing errors without introducing bias to read counts. Sequences were separated according to the individual immunoglobulin specific primers (TAK_402, TAK_403, TAK_IgM, TAK_IgA) and VDJ annotation was conducted with the IMGT/HighV-QUEST service (Alamyar et al., 2012). Sequences using the IgM, IgG1/2 or IgA specific primer were further analyzed with ARGalaxy immune receptor pipeline (IJspeert et al., 2017). Folder contains final data analyzed by ARGalaxy本研究探讨了BXSB小鼠系统性红斑狼疮（Systemic Lupus Erythematosus, SLE）临床前期的短期B细胞耗竭是否会影响其B细胞受体库（B cell receptor repertoire）。研究证实，经抗CD20单克隆抗体（anti-CD20 monoclonal antibody）处理的雄性BXSB小鼠，其IgG VDJ重组的多样性显著降低。 为完成本次分析，本研究依托Illumina MiSeq平台开展二代测序（Next Generation Sequencing, NGS），采用重叠双端读段（2×300 bp）。添加20%的PhiX对照以优化测序性能。双端读段通过IgRepertoireConstructor工具（Safonova等，2015年）进行拼接。测序读段的质量通过MultiQC（Ewels等，2016年）整合FASTQC质量分析结果完成质控。 本研究借助互补DNA（complementary DNA, cDNA）合成环节中的独特分子标识符（Unique Molecular Identifiers, UMI）与多重引物，实现了分子扩增指纹（Molecular Amplification Fingerprinting, MAF）的错误校正。通过模式匹配与筛选包含双UMI的序列，对PCR测序错误进行校正，且不会对读段计数引入偏倚。 根据特异性免疫球蛋白引物（TAK_402、TAK_403、TAK_IgM、TAK_IgA）对序列进行分组，并依托IMGT/HighV-QUEST工具（Alamyar等，2012年）完成VDJ注释。针对使用IgM、IgG1/2或IgA特异性引物的序列，进一步通过ARGalaxy免疫受体分析流程（IJspeert等，2017年）开展分析。 本文件夹包含经ARGalaxy分析得到的最终数据集。