Shallow shotgun metagenome sequencing (SSMS) involves sequencing samples at a shallower depth than is applied in full shotgun metagenome sequencing. By combining many more samples into a single sequencing run and using a modified protocol that uses a lower volume of reagents for sequencing library preparation, SSMS is an economical way to provide compositional and functional sequencing data similar to deep shotgun metagenome sequencing.

Compared to amplicon sequencing, SSMS provides a less biased representation of the microbial community and higher taxonomic resolution; however, it is not a replacement for a full metagenomic study. The cost of SSMS is moderately higher than that of amplicon sequencing but substantially less than that of deep shotgun metagenome sequencing.


To validate our experimental approach, we did not assume that bioinformatically downsampling the number of reads post-sequencing is a proxy for downsampling from data already sequenced. Instead, we multiplexed the samples on a single NexSeq run based on the expected number of reads. This strategy allowed for selectively targeting ~500K to ~2M reads per library after sequencing.

DNA extraction and quality control

DNA is extracted from samples using the Qiagen MagAttract PowerSoil DNA KF Kit (formerly MO BIO PowerSoil DNA Kit) optimized for the Thermofisher KingFisher robot. This kit uses magnetic beads to specifically capture DNA while excluding organic inhibitors. In-house testing has shown that this kit has a good balance of DNA yield and quality, as demonstrated on a variety of environmental sample types.

Library preparation

Whole DNA is prepared for sequencing using the Illumina Nextera Flex DNA library prep kit. Samples are barcoded and mixed together for sequencing. The mix is then subjected to paired-end sequencing using Illumina sequencers.