Comprehensive evaluation and optimization of amplicon library preparation methods for high-throughput antibody...

Area of Science:

• Immunology
• Genomics
• Biotechnology

Background:

• High-throughput sequencing (HTS) of antibody repertoires is vital for systems immunology.
• Potential biases in HTS workflows can impact antibody repertoire data quality.
• The method of adapter addition during library preparation is a critical, yet understudied, factor.

Purpose of the Study:

• To compare the impact of three standard adapter addition methods on antibody repertoire HTS datasets.
• To evaluate the experimental efficiency and practicality of different library preparation techniques.
• To establish a robust protocol for antibody repertoire library generation from low RNA input.

Main Methods:

• Illumina HTS was performed on antibody variable heavy genes from murine antibody-secreting cells.
• Three distinct methods for nucleotide adapter addition were compared.
• Clonal overlap and rank statistics were used to analyze HTS dataset equivalence.

Main Results:

• All investigated adapter addition methods yielded equivalent HTS datasets based on clonal overlap and rank statistics.
• PCR-based methods demonstrated superior speed, efficiency, and practicality compared to ligation-based methods.
• A two-step PCR protocol was developed, enabling library generation from as little as 1 ng of total RNA.

Conclusions:

• The choice of adapter addition method significantly impacts experimental workflow efficiency but not HTS dataset equivalence.
• PCR-based methods are recommended for antibody repertoire library preparation due to their practical advantages.
• This study advances standardized antibody HTS, facilitating systems-based, cross-experiment meta-analyses.