Abstract
High-throughput sequencing (HTS) of antibody repertoire libraries has become a powerful tool in the field of systems immunology. However, numerous sources of bias in HTS workflows may affect the obtained antibody repertoire data. A crucial step in antibody library preparation is the addition of short platform-specific nucleotide adapter sequences. As of yet, the impact of the method of adapter addition on experimental library preparation and the resulting antibody repertoire HTS datasets has not been thoroughly investigated. Therefore, we compared three standard library preparation methods by performing Illumina HTS on antibody variable heavy genes from murine antibody-secreting cells. Clonal overlap and rank statistics demonstrated that the investigated methods produced equivalent HTS datasets. PCR-based methods were experimentally superior to ligation with respect to speed, efficiency, and practicality. Finally, using a two-step PCR based method we established a protocol for antibody repertoire library generation, beginning from inputs as low as 1 ng of total RNA. In summary, this study represents a major advance towards a standardized experimental framework for antibody HTS, thus opening up the potential for systems-based, cross-experiment meta-analyses of antibody repertoires.
Highlights
High-throughput sequencing (HTS) of antibody repertoires offers the potential to study the humoral immune system in a quantitative and systems-based approach [1,2,3,4]
For all library preparation methods, we used an antibody variable heavy chain gene-specific primer set that binds to the first 20–22 nucleotides of framework region 1 (FR1, Table S3 in File S1) [28]. This ensured that differences among HTS datasets were solely attributable to the method of adapter addition used
We presented a comprehensive evaluation of two PCR (DA, PE)- and one ligation-based method for the addition of Illumina sequencing adapters to antibody variable gene libraries examining the effects with respect to yield, practicality, and effect on resulting HTS datasets
Summary
High-throughput sequencing (HTS) of antibody repertoires offers the potential to study the humoral immune system in a quantitative and systems-based approach [1,2,3,4]. Preceding HTS are many experimental steps in the multi-component library preparation, which are prone to biases and errors, and may substantially decrease the accuracy of the HTS delivered antibody repertoire [5]. These biases and errors are related to choice of nucleic acid material [6], PCR protocol variations [7,8,9,10,11,12,13,14], primers needed for specific amplification of antibody genes [15,16], and multiplexed barcoding [17,18]. PCR-based methods have been introduced for adapter addition [15,25,26] in either a one-step (direct addition, DA) or two-step (primer extension, PE) PCR reaction (Fig. 1)
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