Rettig Trisha A, Pecaut Michael J, Chapes Stephen K
Division of Biology, Kansas State University, Manhattan, KS, USA.
Division of Radiation Research, Loma Linda University, Loma Linda, CA, USA.
FASEB Bioadv. 2019 Jan;1(1):6-17. Epub 2018 Aug 30.
Sequencing antibody repertoires has steadily become cheaper and easier. Sequencing methods usually rely on some form of amplification, often a massively multiplexed PCR prior to sequencing. To eliminate potential biases and create a data set that could be used for other studies, our lab compared unamplified sequencing results from the splenic heavy-chain repertoire in the mouse to those processed through two commercial applications. We also compared the use of mRNA vs total RNA, reverse transcriptase, and primer usage for cDNA synthesis and submission. The use of mRNA for cDNA synthesis resulted in higher read counts but reverse transcriptase and primer usage had no statistical effects on read count. Although most of the amplified data sets contained more antibody reads than the unamplified data set, we detected more unique V-gene segments in the unamplified data set. Although unique CDR3 detection was much lower in the unamplified data set, RNASeq detected 98% of the high frequency CDR3s. We have shown that unamplified profiling of the antibody repertoire is possible, detects more V-gene segments, and detects high frequency clones in the repertoire.
对抗体库进行测序已逐渐变得更便宜、更简便。测序方法通常依赖某种形式的扩增，测序前往往是大规模多重PCR。为消除潜在偏差并创建可用于其他研究的数据集，我们实验室将小鼠脾脏重链库的未扩增测序结果与通过两种商业应用程序处理的结果进行了比较。我们还比较了用于cDNA合成和提交的mRNA与总RNA、逆转录酶及引物的使用情况。使用mRNA进行cDNA合成可获得更高的读数，但逆转录酶和引物的使用对读数没有统计学影响。尽管大多数扩增数据集包含的抗体读数比未扩增数据集多，但我们在未扩增数据集中检测到了更多独特的V基因片段。虽然未扩增数据集中独特CDR3的检测率低得多，但RNA测序检测到了98%的高频CDR3。我们已经证明，对抗体库进行未扩增分析是可行的，能够检测到更多的V基因片段，并能检测到库中的高频克隆。
