Development of a closed-tube PCR barcoding strategy to generate highly multiplexed, sequencing ready Illumina libraries with decreased amount of phantom sequences, such as chimeric PCR artifacts.

We present an improved version of our genotyped functional screening platform for high-throughput binder protein screening. By implementing a closed-tube barcoding PCR workflow with 96 + 96 unique and universal indexing primers, we drastically improved sequence clarity, facilitating the identification of true sample sequences and reducing the risk of sample cross-contamination. The system's closed-tube reaction allows for target amplification and indexing using primers with different annealing temperatures (>10 °C), streamlining the process. This improvement enhanced the top-to-second sequence count ratio by threefold and decreased background sequences from 72% to 43% of total sequences. Sample cross-contamination (or index hopping) dropped from 14% to negligible levels, and chimera formation was reduced nearly sixfold. Additionally, by splitting the sequencing adapters between target and indexing primers, the closed-tube method produces sequencing-ready Illumina libraries with fewer phantom sequences. This method is particularly beneficial for low-diversity libraries, such as synthetic antibody libraries, where phantom sequences and chimeras are a common issue due to the high sequence similarity among templates. The reduction in these artifacts ensures more accurate results, improving the reliability of downstream analyses like diversity or enrichment calculations and allows higher number of multiplexed samples. Furthermore, the platform is adaptable to novel binder scaffolds, such as nanobodies or DARPins, by designing two new target amplification primers.