Asset Tag: Shotgun profiling

Recent work comparing metagenomic sequencing methods indicates that a comprehensive picture of the taxonomic and functional diversity of complex communities will be difficult to achieve with one sequencing technology alone. While the lower cost of short reads has enabled greater sequencing depth, the greater contiguity of long-read assemblies and lack of GC bias in SMRT Sequencing has enabled better gene finding. However, since long-read assembly typically requires high coverage for error correction, these benefits have in the past been lost for low-abundance species. The introduction of the Sequel II System has enabled a new, higher throughput, assembly-optional data type that addresses these challenges: HiFi reads. HiFi reads combine QV20 accuracy with long read lengths, eliminating the need for assembly for most metagenome applications, including gene discovery and metabolic pathway reconstruction. In fact, the read lengths and accuracy of HiFi data match or outperform the quality metrics of most metagenome assemblies, enabling cost-effective recovery of intact genes and operons while omitting the resource intensive and data-inefficient assembly step. Here we present the application of HiFi sequencing to both mock and human fecal samples using full-length 16S and shotgun methods. This proof-of-concept work demonstrates the unique strengths of the HiFi method. First, the high correspondence between the expected community composition,16S and shotgun profiling data reflects low context bias. In addition, every HiFi read yields ~5-8 predicted genes, without assembly, using standard tools. If assembly is desired, excellent results can be achieved with Canu and contig binning tools. In summary, HiFi sequencing is a new, cost-effective option for high-resolution functional profiling of metagenomes which complements existing short read workflows.