Finding the right fit: evaluation of short-read and long-read sequencing approaches to maximize the utility of clinical microbiome data

Abstract

A long-standing challenge in human microbiome research is achieving the taxonomic and functional resolution needed to generate testable hypotheses about the gut microbiota’s impact on health and disease. With a growing number of live microbial interventions in clinical development, this challenge is renewed by a need to understand the pharmacokinetics and pharmacodynamics of therapeutic candidates. While short-read sequencing of the bacterial 16S rRNA gene has been the standard for microbiota profiling, recent improvements in the fidelity of long-read sequencing underscores the need for a re-evaluation of the value of distinct microbiome-sequencing approaches. We leveraged samples from participants enrolled in a phase 1b clinical trial of a novel live biotherapeutic product to perform a comparative analysis of short-read and long-read amplicon and metagenomic sequencing approaches to assess their utility for generating clinical microbiome data. Across all methods, overall community taxonomic profiles were comparable and relationships between samples were conserved. Comparison of ubiquitous short-read 16S rRNA amplicon profiling to long-read profiling of the 16S-ITS-23S rRNA amplicon showed that only the latter provided strain-level community resolution and insight into novel taxa. All methods identified an active ingredient strain in treated study participants, though detection confidence was higher for long-read methods. Read coverage from both metagenomic methods provided evidence of active-ingredient strain replication in some treated participants. Compared to short-read metagenomics, approximately twice the proportion of long reads were assigned functional annotations. Finally, compositionally similar bacterial metagenome-assembled genomes (MAGs) were recovered from short-read and long-read metagenomic methods, although a greater number and more complete MAGs were recovered from long reads. Despite higher costs, both amplicon and metagenomic long-read approaches yielded added microbiome data value in the form of higher confidence taxonomic and functional resolution and improved recovery of microbial genomes compared to traditional short-read methodologies.

The Application of Metagenomics to Study Microbial Communities and Develop Desirable Traits in Fermented Foods

“…4300 bp in size) consisting of the 16S rRNA gene, ITS region and 23S rRNA gene [ 117 ]. Targeting the combined 16S-ITS-23S regions instead of individual rRNA locus-derived fragments as commonly done in short read metabarcoding, can provide information on 16S and 23S gene sequences from single reads allowing strain level resolution of microbial communities, and improve diversity, divergence and phylogenetic estimations [ 117 , 118 , 119 , 120 , 121 ]. Depending on the primers used, sequencing of the RRN operon also enables identification and classification of Archaea and Bacteria from the same libraries [ 122 ].…”

Comparing genomes recovered from time-series metagenomes using long- and short-read sequencing technologies

“…The current stage of algorithms and approaches for LR metagenomics is still limited compared to the large toolbox of SR technologies. While the methodology used here re ects the most appropriate tools and algorithms available at the time, we recommend that future studies pursue a critical assessment of newer approaches [12] when using LR techniques. The dataset presented here can also serve as a reference for testing and comparing algorithms and approaches for shotgun LR metagenomic sequencing.…”

“…For instance, current PacBio Sequel II technology offers circular consensus sequencing (CCS), providing a low-error rate in high delity reads, although at a shorter read length than the traditional long-read technology [3]. Additionally, better genome statistics (low number of contigs and high N50 values) [5][6][7][8][9] or the combination of short-and long-reads for the recovery of high-quality MAGs [10,11] have already showcased the bene ts for the recovery of microbial genomes [12]. Nonetheless, whether a switch from SR to LR metagenomic approaches would introduce biases for capturing the genetic potential of microbial populations in terms of recovery of the MAGs is less known.…”

Recovery of Metagenome-Assembled Genomes from a Human Fecal Sample with Pacific Biosciences High-Fidelity Sequencing

“…Recent studies have shown that long-read sequencing technologies improve the contiguity of metagenomic assemblies and enable the recovery of repeated regions compared with short-read alternatives ( 1 , 2 ). To assess long-read technologies and evaluate dedicated bioinformatics tools, we performed deep sequencing of a human fecal metagenome with the PacBio Sequel II system.…”