bioRxiv2024
Metagenomics enables direct investigation of the gene content and potential functions of gut bacteria without isolation and culture. However, metagenome-assembled genomes are often incomplete and have low contiguity due to challenges in assembling repeated genomic elements. Long-read sequencing approaches have successfully yielded circular bacterial genomes directly from metagenomes, but these approaches are low throughput, have high DNA input requirements, and can have high error rates. Illumina has recently launched the Illumina Complete Long Read (ICLR) assay, a new approach for generating kilobase-scale reads with low DNA input requirements and high accuracy. Here, we evaluate the performance of ICLR sequencing for gut metagenomics. We sequenced a microbial mock community and ten human gut microbiome samples with standard, shotgun 2x150 paired-end sequencing, ICLR sequencing, and nanopore long-read sequencing and compared performance in read lengths, assembly contiguity, and bin quality. We find that ICLR human metagenomic assemblies have higher N50 (119.5 {+/-} 24.8 kilobases) than short read assemblies (9.9 {+/-} 4.5 kilobases; p = 0.002), and comparable N50 to nanopore assemblies (91.0 {+/-} 43.8 kilobases; p = 0.32). Additionally, we find that ICLR draft microbial genomes are more complete (94.0% {+/-} 20.6%) than nanopore draft genomes (85.9% {+/-} 23.0%; p[≤] 0.001), and that nanopore draft genomes have truncated gene lengths (924.6 {+/-} 114.7 base pairs) relative to ICLR genomes (954.6 {+/-} 71.5 base pairs; p[≤] 0.001). Overall, we find that ICLR sequencing is a promising method for high-throughput and accurate assembly of microbial genomes from gut metagenomes. ImportanceMetagenomic sequencing allows scientists to directly measure the genome content and structure of microbes residing in complex microbial communities. Traditional short-read metagenomic sequencing methods often yield fragmented genomes, whereas advanced long-read sequencing methods improve genome assembly quality but often suffer from high error rates and are logistically limited due to low throughput and high input requirements. A new method, the Illumina Complete Long Read Assay (ICLR), is capable of generating highly-accurate kilobase-scale sequencing reads with minimal input material. To evaluate the utility of ICLR in metagenomic contexts, we applied short-read, long-read, and ICLR methods to simple and complex microbial communities. We found that ICLR outperforms short-read methods, and yields comparable metagenomic assemblies to standard long-read approaches while requiring less input material. Overall, ICLR represents a high-throughput and efficient solution for assembling complete genomes from complex metagenomes.