Long read-based <i>de novo</i> assembly of low complex metagenome samples results in finished genomes and reveals insights into strain diversity and an active phage system

Somerville, Vincent; Lutz, Stefanie; Schmid, Michael; Frei, Daniel; Moser, Aline; Irmler, Stefan; Frey, Juerg E.; Ahrens, Christian H.

doi:10.1101/476747

Cited by 16 publications

(15 citation statements)

References 98 publications

(112 reference statements)

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“…In contrast, single-molecule sequencing platforms such as the Oxford Nanopore MinION, GridION and PromethION are able to sequence very long fragments of DNA (>10 Kbp, with over 2 Mbp reported) [4,5] and with recent improvements to the platform making metagenomic studies using nanopore more viable, such studies are increasing in frequency [6,7,8,9]. Long reads help with alignment-based assignment of taxonomy and function due to their increased information content [10,11].…”

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confidence: 99%

Ultra-deep, long-read nanopore sequencing of mock microbial community standards

Nicholls

Quick

Tang³

et al. 2018

Preprint

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Background: Long sequencing reads are information-rich: aiding de novo assembly and reference mapping, and consequently have great potential for the study of microbial communities. However, the best approaches for analysis of long-read metagenomic data are unknown. Additionally, rigorous evaluation of bioinformatics tools is hindered by a lack of long-read data from validated samples with known composition. Methods: We sequenced two commercially-available mock communities containing ten microbial species (ZymoBIOMICS Microbial Community Standards) with Oxford Nanopore GridION and PromethION. Isolates from the same mock community were sequenced individually with Illumina HiSeq. Data: We generated 14 and 16 Gbp from GridION owcells and 146 and 148 Gbp from PromethION owcells for the even and odd communities respectively. Read length N50 was 5.3 Kbp and 5.2 Kbp for the even and log community, respectively. Basecalls and corresponding signal data are made available (4.2 TB in total). Results: Alignment to Illumina-sequenced isolates demonstrated the expected microbial species at anticipated abundances, with the limit of detection for the lowest abundance species below 50 cells (GridION). De novo assembly of metagenomes recovered long contiguous sequences without the need for pre-processing techniques such as binning. Conclusions: We present ultra-deep, long-read nanopore datasets from a well-de ned mock community. These datasets will be useful for those developing bioinformatics methods for long-read metagenomics and for the validation and comparison of current laboratory and software pipelines.Whole-genome sequencing of microbial communities (metagenomics) has revolutionised our view of microbial evolution and diversity, with numerous potential applications for microbial ecology, clinical microbiology and industrial biotechnology [1,2]. Typically, metagenomic studies use high-throughput sequencing platforms (e.g. Illumina) [3] which generate very high yield, but of limited read length (100-300 bp).

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confidence: 99%

Ultra-deep, long-read nanopore sequencing of mock microbial community standards

Nicholls

Quick

Tang³

et al. 2018

Preprint

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“…Wick and Holt, 2019 recently benchmarked various long-read assemblers and demonstrated that Flye improves on other long-read assemblers in the case of bacterial genomes. Since this conclusion was further confirmed in recent bacterial studies (Ring et al, 2018, Schmid et al, 2018, Somerville et al, 2019, we only analyzed how mosaicFlye improves on the Flye and metaFlye (Kolmogorov et al, 2019b) assemblies in the case of bacterial genomes and metagenomes. In the case of the human genome, we analyzed both Canu and Flye assemblies.…”

Section: Resultsmentioning

confidence: 66%

mosaicFlye: Resolving long mosaic repeats using long error-prone reads

Pevzner

2020

Preprint

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Long-read technologies revolutionized genome assembly and enabled resolution of bridged repeats (i.e., repeats that are spanned by some reads) in various genomes. However, the problem of resolving unbridged repeats (such as long segmental duplications in the human genome) remains largely unsolved, making it a major obstacle towards achieving the goal of complete genome assemblies. Moreover, the challenge of resolving unbridged repeats is not limited to eukaryotic genomes but also impairs assemblies of bacterial genomes and metagenomes. We describe the mosaicFlye algorithm for resolving complex unbridged repeats based on differences between various repeat copies and show how it improves assemblies of the human genome as well as bacterial genomes and metagenomes. In particular, we show that mosaicFlye results in a complete assembly of both arms of the human chromosome 6.

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“…Some of the limitations to obtain complete genomes using high-throughput sequencing (HTS) are mainly due to the shorter reads produced. Short reads not only can impair the detection of different viral haplotypes (Takeda et al, 2019) and introduce ambiguities in de novo assemblies (Schatz et al, 2010) but are also unable to resolve complex regions of the genome (Somerville et al, 2019;Treangen and Salzberg, 2011). Additionally, the PCR amplification steps involved during library preparation can introduce considerable bias, for example selective amplification and chimeras (Ardui et al, 2018;Ng et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

Whole Genome Sequencing of Hepatitis A Virus Using a PCR-Free Single-Molecule Nanopore Sequencing Approach

et al. 2020

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Hepatitis A virus (HAV) is one of the most common causes of acute viral hepatitis in humans. Although HAV has a relatively small genome, there are several factors limiting whole genome sequencing such as PCR amplification artefacts and ambiguities in de novo assembly. The recently developed Oxford Nanopore technologies (ONT) allows single-molecule sequencing of long-size fragments of DNA or RNA using PCR-free strategies. We have sequenced the whole genome of HAV using a PCR-free approach by direct reverse-transcribed sequencing. We were able to sequence HAV cDNA and obtain reads over 7 kilobases in length containing almost the whole genome of the virus. The comparison of these raw long nanopore reads with the HAV reference wild type revealed a nucleotide sequence identity between 81.1 and 96.6%. By de novo assembly of all HAV reads we obtained a consensus sequence of 7362 bases, with a nucleotide sequence identity of 99.0% with the genome of the HAV strain pHM175/18f. When the assembly was performed using as reference the HAV strain pHM175/18f a consensus with a sequence similarity of 99.8 % was obtained. We have also used an ONT amplicon-based assay to sequence two fragments of the VP3 and VP1 regions which showed a sequence similarity of 100% with matching regions of the consensus sequence obtained using the direct cDNA sequencing approach. This study showed the applicability of ONT sequencing technologies to obtain the whole genome of HAV by direct cDNA nanopore sequencing, highlighting the utility of this PCR-free approach for HAV characterization and potentially other viruses of the Picornaviridae family.

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Long read-based de novo assembly of low complex metagenome samples results in finished genomes and reveals insights into strain diversity and an active phage system

Cited by 16 publications

References 98 publications

Ultra-deep, long-read nanopore sequencing of mock microbial community standards

Ultra-deep, long-read nanopore sequencing of mock microbial community standards

mosaicFlye: Resolving long mosaic repeats using long error-prone reads

Whole Genome Sequencing of Hepatitis A Virus Using a PCR-Free Single-Molecule Nanopore Sequencing Approach

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