Functional meta-omics provide critical insights into long- and short-read assemblies

Galata, Valentina; Busi, Susheel Bhanu; Kunath, Benoît J.; Nies, Laura de; Całusińska, Magdalena; Halder, Rashi; Wilmes, Paul; Laczny, Cédric Christian

doi:10.1093/bib/bbab330

Cited by 8 publications

(5 citation statements)

References 48 publications

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“…In this study, we report how lower than recommended DNA input affects Nanopore long-read metagenomics by comparing different levels of metagenomic analysis from read QC to retrieval of MAGs. Many previously published Nanopore-based metagenomics studies lack information about the quantity of DNA used for library preparation [19,[24][25][26]32]. To the best of our knowledge, other published metagenomic studies quantified the amount of input DNA and met or exceeded the recommended amount of 1000 ng DNA for input material.…”

Section: Discussionmentioning

confidence: 99%

Dancing the Nanopore limbo – Nanopore metagenomics from small DNA quantities for bacterial genome reconstruction

Simon

Schmidt

Griesdorn

et al. 2023

Preprint

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Background: While genome-resolved metagenomics has revolutionized our understanding of microbial and genetic diversity in environmental samples, assemblies of short-reads often result in incomplete and/or highly fragmented metagenome-assembled genomes (MAGs), hampering in-depth genomics. Although Nanopore sequencing has increasingly been used in microbial metagenomics as long reads greatly improve the assembly quality of MAGs, the recommended DNA quantity usually exceeds the recoverable amount of DNA of environmental samples. Here, we evaluated lower-than-recommended DNA quantities for Nanopore library preparation by determining sequencing quality, community composition, assembly quality and recovery of MAGs. Results: We generated 27 Nanopore metagenomes using the commercially available ZYMO mock community and varied the amount of input DNA from 1000 ng (the recommended minimum) down to 1 ng in eight steps. The quality of the generated reads remained stable across all input levels. The read mapping accuracy, which reflects how well the reads match a known reference genome, was consistently high across all libraries. The relative abundance of the species in the metagenomes was stable down to input levels of 50 ng. High-quality MAGs (> 95% completeness, ≤ 5% contamination) could be recovered from metagenomes down to 35 ng of input material. When combined with publicly available Illumina reads for the mock community, Nanopore reads from input quantities as low as 1 ng improved the quality of hybrid assemblies. Conclusion: Our results show that the recommended DNA amount for Nanopore library preparation can be substantially reduced without any adverse effects to genome recovery and still bolster hybrid assemblies when combined with short-read data. We posit that the results presented herein will enable studies to improve genome recovery from low-biomass environments, enhancing microbiome understanding.

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Section: Discussionmentioning

confidence: 99%

Dancing the Nanopore limbo – Nanopore metagenomics from small DNA quantities for bacterial genome reconstruction

Simon

Schmidt

Griesdorn

et al. 2023

Preprint

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“…Brown et al (2021) used resistome risk score based on co-occurrences of ARGs, MGEs and pathogen gene markers on the same contig to evaluate convergence of biological output produced by different assemblers. Another paper by Galata et al (2021) showed that the choices of assembly software as well as sample complexity have considerable impact on prediction of genes and proteins. A recent study by Yorki et al (2023) focused on assessment of short-, long- and hybrid-approaches to recover the genome of clinically relevant low-abundant E. coli and their ARG content from metagenomic samples.…”

Section: Discussionmentioning

confidence: 99%

Metagenomic assemblies tend to break around antibiotic resistance genes

Abramova,

Karkman,

Bengtsson-Palme

2023

Preprint

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BackgroundAssembly of metagenomic samples can provide essential information about the mobility potential and taxonomic origin of antibiotic resistance genes (ARGs), and inform interventions to prevent further spread of resistant bacteria. However, ARGs typically occur in multiple genomic contexts across different species, representing a considerable challenge for the assembly process. Usually, this results in many fragmented contigs of unclear origin, complicating the risk assessment of ARG detections. To systematically investigate the impact of this issue on detection, quantification and contextualization of ARGs, we evaluated the performance of different assembly approaches, including genomic-, metagenomic- and transcriptomic-specialized assemblers. We quantified recovery and accuracy rates of each tool for ARGs both fromin silicospiked metagenomic samples as well as real samples sequenced using both long- and short-read sequencing technologies.ResultsThe results revealed that none of the investigated tools can accurately capture genomic contexts present in samples of high complexity. The transcriptomic assembler Trinity showed a better performance in terms of reconstructing longer and fewer contigs matching unique genomic contexts, which can be beneficial for deciphering the taxonomic origin of ARGs. The currently commonly used metagenomic assembly tools metaSPAdes and MEGAHIT were able to identify the ARG repertoire but failed to fully recover the diversity of genomic contexts present in a sample. On top of that, in a complex scenario MEGAHIT produced very short contigs, which can lead to considerable underestimation of the resistome in a given sample.ConclusionsOur study shows that metaSPAdes and Trinity would be the preferable tools in terms of accuracy to recover correct genomic contexts around ARGs in metagenomic samples characterized by uneven coverages. Overall, the inability of assemblers to reconstruct long ARG-containing contigs has impacts on ARG quantification, suggesting that directly mapping reads to an ARG database might be necessary, at least as a complementary strategy, to get accurate ARG abundance and diversity measures.

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“…In this study, we report how lower than recommended DNA input affects Nanopore long-read metagenomics by comparing different levels of metagenomic analysis from read QC to retrieval of MAGs. Many previously published Nanopore-based metagenomics studies lack information about the quantity of DNA used for library preparation [ 19 , 24 – 26 , 32 ]. To the best of our knowledge, other published metagenomic studies quantified the amount of input DNA and met or exceeded the recommended amount of 1000 ng DNA for input material.…”

Section: Discussionmentioning

confidence: 99%

Dancing the Nanopore limbo – Nanopore metagenomics from small DNA quantities for bacterial genome reconstruction

Simon,

Schmidt,

Griesdorn

et al. 2023

BMC Genomics

View full text Add to dashboard Cite

Background While genome-resolved metagenomics has revolutionized our understanding of microbial and genetic diversity in environmental samples, assemblies of short-reads often result in incomplete and/or highly fragmented metagenome-assembled genomes (MAGs), hampering in-depth genomics. Although Nanopore sequencing has increasingly been used in microbial metagenomics as long reads greatly improve the assembly quality of MAGs, the recommended DNA quantity usually exceeds the recoverable amount of DNA of environmental samples. Here, we evaluated lower-than-recommended DNA quantities for Nanopore library preparation by determining sequencing quality, community composition, assembly quality and recovery of MAGs. Results We generated 27 Nanopore metagenomes using the commercially available ZYMO mock community and varied the amount of input DNA from 1000 ng (the recommended minimum) down to 1 ng in eight steps. The quality of the generated reads remained stable across all input levels. The read mapping accuracy, which reflects how well the reads match a known reference genome, was consistently high across all libraries. The relative abundance of the species in the metagenomes was stable down to input levels of 50 ng. High-quality MAGs (> 95% completeness, ≤ 5% contamination) could be recovered from metagenomes down to 35 ng of input material. When combined with publicly available Illumina reads for the mock community, Nanopore reads from input quantities as low as 1 ng improved the quality of hybrid assemblies. Conclusion Our results show that the recommended DNA amount for Nanopore library preparation can be substantially reduced without any adverse effects to genome recovery and still bolster hybrid assemblies when combined with short-read data. We posit that the results presented herein will enable studies to improve genome recovery from low-biomass environments, enhancing microbiome understanding.

show abstract

Functional meta-omics provide critical insights into long- and short-read assemblies

Cited by 8 publications

References 48 publications

Dancing the Nanopore limbo – Nanopore metagenomics from small DNA quantities for bacterial genome reconstruction

Dancing the Nanopore limbo – Nanopore metagenomics from small DNA quantities for bacterial genome reconstruction

Metagenomic assemblies tend to break around antibiotic resistance genes

Dancing the Nanopore limbo – Nanopore metagenomics from small DNA quantities for bacterial genome reconstruction

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