Validation of Methods to Assess the Immunoglobulin Gene Repertoire in Tissues Obtained from Mice on the International Space Station

Spaceflight is known to affect immune cell populations. In particular, splenic B-cell numbers decrease during spaceflight and in ground-based physiological models. Although antibody isotype changes have been assessed during and after spaceflight, an extensive characterization of the impact of spaceflight on antibody composition has not been conducted in mice. Next Generation Sequencing and bioinformatic tools are now available to assess antibody repertoires. We can now identify immunoglobulin gene- segment usage, junctional regions, and modifications that contribute to specificity and diversity. Due to limitations on the International Space Station, alternate sample collection and storage methods must be employed. Our group compared Illumina MiSeq sequencing data from multiple sample preparation methods in normal C57Bl/6J mice to validate that sample preparation and storage would not bias the outcome of antibody repertoire characterization. In this report, we also compared sequencing techniques and a bioinformatic workflow on the data output when we assessed the IgH and Igκ variable gene usage. Our bioinformatic workflow has been optimized for Illumina HiSeq and MiSeq datasets, and is designed specifically to reduce bias, capture the most information from Ig sequences, and produce a data set that provides other data mining options.

已知太空飞行会对免疫细胞群产生影响。具体而言，在太空飞行期间以及地面模拟生理模型中，脾脏B细胞数量均会出现下降。尽管已有研究在太空飞行期间及飞行后对抗体同种型（antibody isotype）的变化进行了评估，但目前尚未在小鼠模型中全面表征太空飞行对抗体组成的影响。如今，下一代测序（Next Generation Sequencing）与生物信息学工具已可用于抗体库（antibody repertoire）的评估工作，我们能够借此鉴定免疫球蛋白基因片段的使用情况、连接区序列以及影响抗体特异性与多样性的各类修饰。由于国际空间站（International Space Station）存在条件限制，必须采用替代的样本采集与储存方法。本研究团队以正常C57Bl/6J小鼠为对象，对比了多种样本制备方法对应的Illumina MiSeq测序数据，以验证样本制备与储存流程不会对抗体库表征的结果产生偏倚。本报告中，我们还在评估免疫球蛋白重链（Immunoglobulin Heavy Chain, IgH）与免疫球蛋白κ可变基因（Immunoglobulin κ variable gene, Igκ）的使用情况时，对比了不同测序技术及一套生物信息学工作流程对数据产出的影响。本团队搭建的生物信息学工作流程已针对Illumina HiSeq与MiSeq测序数据集完成优化，其专门设计用于降低分析偏倚、最大化捕获免疫球蛋白序列信息，并生成可支持多维度数据挖掘的数据集。