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 faecal 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, but 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 tradeoff between the quantity and quality of recovered genomes.ImportanceOur understanding of microbial communities is limited by the technologies we employ. Here, we test several different DNA sequencing techniques to better understand the pros and cons of each. Long read DNA sequencing allowed for the reconstruction of higher quality and even complete microbial genomes, however, the cost was greater than commonly used short-read DNA sequencing. We suggest researchers consider the trade-offs between each method and decide based on the goals of their research question/s.

Highly accurate metagenome-assembled genomes from human gut microbiota using long-read assembly, binning, and consolidation methods

“…PacBio HiFi sequencing produces highly accurate consensus reads (>Q20, median Q30) that are 10–20 kb in length (Wenger et al 2019). Several studies have demonstrated that HiFi sequencing generally produces more total MAGs and higher quality MAGs than short-read sequencing (Priest et al 2021; Gehrig et al 2022; Meslier et al 2022; Eisenhofer et al 2023; Orellana et al 2023; Tao et al 2023; Zhang et al 2023), and that HiFi sequencing outperforms ONT for metagenome assembly (Meslier et al 2022; Sereika et al 2022). The high accuracy of HiFi reads has led to the development of two new metagenome assembly methods, hifiasm-meta (Feng et al 2022) and metaMDBG (Benoit et al 2023), which perform better than previous methods such as HiCanu (Nurk et al 2020) and metaFlye (Kolmogorov et al 2020).…”

Roadmap for the integration of environmental microbiomes in risk assessments under EFSA's remit

“…in 2023 demonstrated the usefulness of combining both technologies thanks to their respective strengths (Eisenhofer et al, 2023;Orellana et al, 2023;Wick et al, 2023). In addition, the rapid evolution in single-cell sequencing promises to open new perspectives and deepen even further our knowledge of environmental microbiomes (Lloréns-Rico et al, 2022).…”