Advancements in next-generation sequencing (NGS) continue to decrease sequencing costs by increasing data generated per run. This reduction in sequencing cost enables population-level genomic experiments to help study and diagnose genetic disorders. Despite sequencer advancements, efficient NGS library preparation is hindered by the cost and effort needed to process individual samples. Libraries need to be quantified and pooled to equitably distribute sequencing reads, which becomes prohibitively tedious, costly, and time-consuming as sample numbers increase. We present a streamlined NGS library preparation technology that eliminates the need for sample-by-sample handling normalization, and pools samples early in the process, resulting in great simplification in workflow and up to a 48x increase in post-ligation sample processing throughput without compromising data quality.

This high-throughput workflow is built upon enzymatic fragmentation of samples that are converted to sequenceable libraries using novel normalization adapters with inline barcodes. Library normalization occurs from adapters during the ligation step, achieving library conversion independent from DNA input mass (20-200 ng, CV < 20%) on-par with qPCR-based pooling. Up to 48 inline barcodes are included on the adapters which uniformly ligate to samples for simple demultiplexing. The design of the inline barcodes allows for flexible pooling where up to 48 samples can be pooled in a modular manner. With normalization adapters and inline barcodes, pooling is performed immediately after adapter ligation, significantly reducing the number of clean-up and PCR reactions. This format can also support up to tens of thousands of libraries in a single sequencing run using dual unique index sequencing primers post-ligation. Finally,
multiplexed PCR amplification artifacts can be reduced using this workflow. Library conversion is consistent and well-suited for both low-pass whole genome sequencing (WGS) and targeted sequencing for variant calling (>20x coverage).

This new library preparation process applies a novel technology to reduce hands-on time and increase efficiency. A process that once treated each sample individually can now be pooled while ensuring equal conversion independent of DNA mass input. This workflow significantly advances the experimental process of library preparation to complement throughput advancements made in sequencing instrumentation.