We present an improved version of DNA indexing platform allowing geno-phenotype analysis of all binder protein clones arrayed for high-throughput screening. The method was optimized for two primer pairs with differing annealing temperatures (>10 °C), resulting in plate and well ID barcoding in a single, closed-tube PCR. As compared to our earlier hierarchical indexing, the closed-tube approach 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) decreased 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 artifact sequences.

This method is particularly beneficial for amplicons with high sequence homology, such as synthetic antibody libraries, where chimeras and other background sequences are commonly encountered in Illumina sequencing with highly multiplexed indexing schemes. The reduction in these artifacts ensures more accurate results, improving the reliability of downstream analyses (e.g. diversity or enrichment calculations) and allows a 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.