Critical evaluation of short, long, and hybrid assembly for contextual analysis of antibiotic resistance genes in complex environmental metagenomes

Abstract

In the fight to limit the global spread of antibiotic resistance, the assembly of environmental metagenomes has the potential to provide rich contextual information (e.g., taxonomic hosts, carriage on mobile genetic elements) about antibiotic resistance genes (ARG) in the environment. However, computational challenges associated with assembly can impact the accuracy of downstream analyses. This work critically evaluates the impact of assembly leveraging short reads, nanopore MinION long-reads, and a combination of the two (hybrid) on ARG contextualization for ten environmental metagenomes using seven prominent assemblers (IDBA-UD, MEGAHIT, Canu, Flye, Opera-MS, metaSpades and HybridSpades). While short-read and hybrid assemblies produced similar patterns of ARG contextualization, raw or assembled long nanopore reads produced distinct patterns. Based on an in-silico spike-in experiment using real and simulated reads, we show that low to intermediate coverage species are more likely to be incorporated into chimeric contigs across all assemblers and sequencing technologies, while more abundant species produce assemblies with a greater frequency of inversions and insertion/deletions (indels). In sum, our analyses support hybrid assembly as a valuable technique for boosting the reliability and accuracy of assembly-based analyses of ARGs and neighboring genes at environmentally-relevant coverages, provided that sufficient short-read sequencing depth is achieved.

Antibiotic resistance: Time of synthesis in a post-genomic age

“…However, even in this case, the incorporation of HGT-acquired genes into the genomes is still cumbersome, unless the genomes containing ARGs are highly represented in the metagenome, because they present a different nucleotide composition and, if present in plasmid a different abundance. Sequence methods that provide long reads can help to solve this problem, and the combination of long- and short-reads information for assembling seems to be superior than using independently either short-reads or long-reads based methodologies, however costs will be higher too [310] . Hybrid methods making use of information on abundance and composition are the best option for binning the generated contigs [311] .…”

Surveillance of potential pathogens and antibiotic resistance in wastewater and surface water from Boston, USA and Vellore, India using long-read metagenomic sequencing

“…It is important to consider that assembler performance varies by coverage and complexity of microbial communities. For example, in an investigation of short, long, and hybrid assemblers for analysis of wastewater metagenomes, metaFlye produced the longest contigs and most co-occurrences of antibiotic resistance genes with mobile genetic elements and pathogen gene markers but was also shown to be vulnerable to misassemblies at low coverages 52 . Canu 44 has also been identified as a promising assembler for long reads but requires a significant amount of computing time and memory, often exceeding that available on university high-performance computer clusters with job time and memory limitations 45,52 .…”

Wastewater Based Epidemiology Enabled Surveillance of Antibiotic Resistance

“…other MGEs were investigated and visualized via network analysis of assembled scaffolds (Figure 5). While acknowledging that there is uncertainty in the accuracy of scaffolds assembled from metagenomic data, [50] we carried out the analysis for empirical comparison assuming a consistent error rate across a sample set generated from the same sequencing platform. A substantial portion of reads were assembled, averaging 34% and ranging from 24% (HKG1-P1) to 50% (PHL1-P2).…”