Implications of Error-Prone Long-Read Whole-Genome Shotgun Sequencing on Characterizing Reference Microbiomes

Hu, Yu; Li, Fang; Nicholson, Christopher; Wang, Kai

doi:10.1016/j.isci.2020.101223

Cited by 16 publications

(17 citation statements)

References 43 publications

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“…S4) 33,46 . RedBean displayed a reduced performance in comparison to other longread assemblers 32,33,46,47 . To the best of our knowledge, no other metagenome assembly benchmark has included Pomoxis, Shasta, or Raven.…”

Section: Discussionmentioning

confidence: 82%

“…Our results are in accordance with previous studies. MetaFlye has proved to outperform other tools in terms of metagenome recovery when using different mock communities 32,33 , although it must be noted that these previous studies did not include all the tools selected in the present benchmark. Canu also performed well in other studies 33 , and has been proposed for increasing the contiguity of metagenome assembled genomes recovered from real samples 46 .…”

Section: Discussionmentioning

confidence: 94%

See 1 more Smart Citation

Assembly methods for nanopore-based metagenomic sequencing: a comparative study

Latorre‐Pérez¹,

Villalba-Bermell²,

Pascual³

et al. 2020

Sci Rep

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Metagenomic sequencing has allowed for the recovery of previously unexplored microbial genomes. Whereas short-read sequencing platforms often result in highly fragmented metagenomes, nanoporebased sequencers could lead to more contiguous assemblies due to their potential to generate long reads. Nevertheless, there is a lack of updated and systematic studies evaluating the performance of different assembly tools on nanopore data. In this study, we have benchmarked the ability of different assemblers to reconstruct two different commercially-available mock communities that have been sequenced using Oxford Nanopore Technologies platforms. Among the tested tools, only metaFlye, Raven, and Canu performed well in all the datasets. These tools retrieved highly contiguous genomes (or even complete genomes) directly from the metagenomic data. Despite the intrinsic high error of nanopore sequencing, final assemblies reached high accuracy (~ 99.5 to 99.8% of consensus accuracy). Polishing strategies demonstrated to be necessary for reducing the number of indels, and this had an impact on the prediction of biosynthetic gene clusters. correction with high quality short reads did not always result in higher quality draft assemblies. Overall, nanopore metagenomic sequencing data-adapted to MinION's current output-proved sufficient for assembling and characterizing lowcomplexity microbial communities. Background Metagenomic sequencing has revolutionized the way we study and characterize microbial communities. This culture-independent technique based on shotgun sequencing has been applied in a broad range of biological fields, ranging from microbial ecology 1 to evolution 2 , or even clinical microbiology 3. In recent years, metagenomics has also become a powerful tool for recovering individual genomes directly from complex microbiomes 2,4,5 , leading to the identification and description of new-and mostly unculturable-taxa with meaningful implications 6. Illumina has been the most widely used platform for metagenomic studies. Illumina reads are characterized by their short length (75-300 bp) and high accuracy (~ 0.1% of basecalling errors) 7. When performing de novo assemblies, Illumina sequences often result in highly fragmented genomes, even when sequencing pure cultures 8,9. This is a consequence of the inability to correctly assemble genomic regions containing repetitive elements that are longer than the read length 9. This fragmentation problem is magnified when handling metagenomic sequences due to the existence of intergenomic repeats that are shared by more than one taxon present in the microbial community 10. It has to be noted that microbial communities often contain related species or sub-species in different-and unknown-abundances, resulting in extensive intergenomic overlaps that can hinder the assembly process 11,12. Third generation sequencing platforms have recently emerged as a solution to resolve ambiguous repetitive regions and to improve genome contiguity. Despite the considerable error associated to these te...

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Section: Discussionmentioning

confidence: 82%

Section: Discussionmentioning

confidence: 94%

Assembly methods for nanopore-based metagenomic sequencing: a comparative study

Latorre‐Pérez¹,

Villalba-Bermell²,

Pascual³

et al. 2020

Sci Rep

View full text Add to dashboard Cite

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“…However substantial limitations of this approach have become evident, including problems related to the use of multi-sample co-assemblies 19 , 66 , 67 , the challenges of resolving genomes to strain level 68 , difficulties related to extracting MAGs from communities of high ecological complexity 69 , 70 and the limitations of automated binning procedures, requiring careful evaluation of recovered genomes 71 . In response to these challenges, recent efforts have combined short read with emerging complementary techniques such as HiC metagenomics 72 – 74 , synthetic long reads 75 , 76 , or long read sequencing 12 , 16 – 23 and collectively these results suggest substantial improvements can be made in the quality and completeness of metagenome-assembled genomes using multiple types of sequence data.…”

Section: Discussionmentioning

confidence: 99%

“…New long read analysis methods 13 , 14 and binning algorithms designed for long read metagenome data 15 have also appeared, anticipating the future expansion of metagenome data generated from these new instruments. More recent studies 12 , 16 – 23 have collectively established that full length (or near-full length genomes) can be recovered from long read sequencing of complex communities, which sets the stage for further development of genome-resolved long read metagenomics.…”

Section: Introductionmentioning

confidence: 99%

Recovery of complete genomes and non-chromosomal replicons from activated sludge enrichment microbial communities with long read metagenome sequencing

Arumugam

Bessarab

Haryono

et al. 2021

npj Biofilms Microbiomes

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New long read sequencing technologies offer huge potential for effective recovery of complete, closed genomes from complex microbial communities. Using long read data (ONT MinION) obtained from an ensemble of activated sludge enrichment bioreactors we recover 22 closed or complete genomes of community members, including several species known to play key functional roles in wastewater bioprocesses, specifically microbes known to exhibit the polyphosphate- and glycogen-accumulating organism phenotypes (namely Candidatus Accumulibacter and Dechloromonas, and Micropruina, Defluviicoccus and Candidatus Contendobacter, respectively), and filamentous bacteria (Thiothrix) associated with the formation and stability of activated sludge flocs. Additionally we demonstrate the recovery of close to 100 circularised plasmids, phages and small microbial genomes from these microbial communities using long read assembled sequence. We describe methods for validating long read assembled genomes using their counterpart short read metagenome-assembled genomes, and assess the influence of different correction procedures on genome quality and predicted gene quality. Our findings establish the feasibility of performing long read metagenome-assembled genome recovery for both chromosomal and non-chromosomal replicons, and demonstrate the value of parallel sampling of moderately complex enrichment communities to obtaining high quality reference genomes of key functional species relevant for wastewater bioprocesses.

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“…Despite the advantages over short-read sequencing data, complete contiguous assemblies are still constrained by the relatively high error rate of nanopore reads, and the quality of the metagenome assembly is related to the coverage of the different species present in the sample, which in turn depends on the experiment throughput. In practice, the read-length advantage of nanopore enables nearly complete assemblies even for low abundance strains, provided that they are covered at a minimal level [89] .…”

Section: Metagenomicsmentioning

confidence: 99%