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 (EMP) 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 (ITS) 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 new 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.