A comparison of short-read, HiFi long-read, and hybrid strategies for genome-resolved metagenomics

Abstract

Shotgun metagenomics enables the reconstruction of complex microbial communities at a high level of detail. Such an approach can be conducted using both short-read and long-read sequencing data, as well as a combination of both. To assess the pros and cons of these different approaches, we used 22 fecal DNA extracts collected weekly for 11 weeks from two respective lab mice to study seven performance metrics over four combinations of sequencing depth and technology: (i) 20 Gbp of Illumina short-read data, (ii) 40 Gbp of short-read data, (iii) 20 Gbp of PacBio HiFi long-read data, and (iv) 40 Gbp of hybrid (20 Gbp of short-read +20 Gbp of long-read) data. No strategy was best for all metrics; instead, each one excelled across different metrics. The long-read approach yielded the best assembly statistics, with the highest N50 and lowest number of contigs. The 40 Gbp short-read approach yielded the highest number of refined bins. Finally, the hybrid approach yielded the longest assemblies and the highest mapping rate to the bacterial genomes. Our results suggest that while long-read sequencing significantly improves the quality of reconstructed bacterial genomes, it is more expensive and requires deeper sequencing than short-read approaches to recover a comparable amount of reconstructed genomes. The most optimal strategy is study-specific and depends on how researchers assess the trade-off between the quantity and quality of recovered genomes. IMPORTANCE Mice are an important model organism for understanding the gut microbiome. When studying these gut microbiomes using DNA techniques, researchers can choose from technologies that use short or long DNA reads. In this study, we perform an extensive benchmark between short- and long-read DNA sequencing for studying mice gut microbiomes. We find that no one approach was best for all metrics and provide information that can help guide researchers in planning their experiments.

The Third-Generation Sequencing Challenge: Novel Insights for the Omic Sciences

“…Nevertheless, PacBio sequencing has been successfully used in several metagenomic studies and was able, for example, to identify the microbiome profiles of heavy-metal-contaminated soils [89], analyze extrachromosomal genetic elements (mainly plasmids) in the human gut [90], characterize the airway microbiome of chronic obstructive pulmonary disease patients [91], identify low-biomass human gut phageomes [92], and highlight the association between the indoor microbiome and nasal/oral humans microbiome [93]. A very recent paper by Eisenhofer and colleagues compared shotgun metagenomics carried out with both NGS and PacBio technologies and found that even if PacBio ameliorates the quality of bacterial genome assembly, it is more expensive and needs a higher sequencing coverage with respect to short reads, thus suggesting that the most optimal strategy depends on the aims of each specific project [94].…”

Seeing in the dark: a metagenomic approach can illuminate the drivers of plant disease

“…Short-read platforms offer depth, capturing rare or low-diversity species and providing comprehensive nucleotide diversity information. In contrast, long-read platforms provide better coverage and are especially beneficial for studying microbial functioning ( Eisenhofer et al., 2024 ). Despite the potential for a lower number of reads with long-read platforms, their extended length affords greater selectivity during read classification.…”