HighALPS: ultra-high-throughput marker-gene amplicon library preparation and sequencing on the Illumina NextSeq and NovaSeq Platforms

Microbiome research using amplicon sequencing of microbial marker genes has surged over the past decade, propelled by protocols for highly multiplexed sequencing with barcoded primer constructs. Newer Illumina platforms like the NovaSeq and NextSeq series significantly outperform older sequencers in terms of reads, output, and runtime. However, these platforms are more prone to index-hopping, which limits the application of protocols designed for older platforms such as the Earth Microbiome Project protocols; hence, there is a need to adapt these established protocols. Here, we present an ultra- high -throughput a mplicon l ibrary p reparation and s equencing protocol (HighALPS) incorporating the capabilities of these newer sequencing platforms, designed for both 16S rRNA gene and fungal internal transcribed spacer domain sequencing. Our results demonstrate good run performance across different sequencing platforms and flow cells, with successful sequencing of mock communities, validating the protocol’s effectiveness. The HighALPS library preparation method offers a robust, cost-effective, and ultra-high-throughput solution for microbiome research, compatible with the latest sequencing technologies. This protocol allows multiplexing thousands of samples in a single run at a read depth of tens of millions of sequences per sample. IMPORTANCE Marker gene amplicon sequencing on Illumina devices remains the most commonly used technology to profile microbial communities. Yet, most library preparation protocols are not adapted to harness the capabilities and deal with the caveats of the latest Illumina sequencing platforms, which highly outperform older platforms in terms of speed, quality, and output. Here, we present an ultra-high-throughput, cost-effective, and robust library preparation protocol (HighALPS) optimized to fully leverage the capabilities of the latest Illumina sequencing platforms. The combinatorial unique dual index strategy effectively combats miss-assignment of reads due to index-hopping, which is more prevalent in newer platforms. The HighALPS protocol incorporates technological (e.g., novel sequencing chemistry and lab automation platforms) as well as bioinformatics advances (e.g., denoising algorithms which make triplicate amplifications unnecessary) of the last few years to optimize and streamline library preparation for bacterial and fungal communities.