Confident phylogenetic identification of uncultured prokaryotes through long read amplicon sequencing of the 16S‐ITS‐23S rRNA operon

Martijn, Joran; Lind, Anne-Li; Schön, Max Emil; Spiertz, Ian; Juzokaite, Lina; Bunikis, Ignas; Pettersson, Olga Vinnere; Ettema, Thijs J. G.

doi:10.1111/1462-2920.14636

Cited by 59 publications

(62 citation statements)

References 53 publications

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“…With our method, it is now possible to effortless improve upon highthroughput sequencing of environmental samples with databases based on full rRNA operon (SSU-ITS-LSU), which hasn't been feasible before due to the length of the operon (≈ 5 kbp) and the method limitations described earlier. A database of full operon rRNA sequences will help improve upon rRNA phylogeny, allow higher phylogenetic resolution [40][41][42][43] , especially critical if the method is applicable to eukaryotes 44,45 , and will present a wider range of target regions for short-read amplicon sequencing and FISH probes 46,47 .…”

Section: Resultsmentioning

confidence: 99%

“…Chimera detection is notoriously difficult when sequencing errors are present and uchime2_ref, which we used, will only call a chimera if a sequence is an error-free combination of the references 40 . We estimate that approximately ~10% of our consensus sequences are error-free, and hence the chimera detection only works as intended on that fraction of the data.…”

Section: Figure S6: Validation Of Chimera Detectionmentioning

confidence: 99%

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Enabling high-accuracy long-read amplicon sequences using unique molecular identifiers with Nanopore or PacBio sequencing

Karst

Ziels

Kirkegaard

et al. 2019

Preprint

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High-throughput amplicon sequencing of large genomic regions represents a challenge for existing short-read technologies. Long-read technologies can in theory sequence large genomic regions, but they currently suffer from high error rates. Here, we report a highthroughput amplicon sequencing approach that combines unique molecular identifiers (UMIs) with Oxford Nanopore sequencing to generate single-molecule consensus sequences of large genomic regions. We demonstrate the approach by generating nearly 10,000 full-length ribosomal RNA (rRNA) operons of roughly 4,400 bp in length from a mock microbial community consisting of eight bacterial species using a single Oxford Nanopore MinION flowcell. The mean error rate of the consensus sequences was 0.03%, with no detectable chimeras due to a rigorous UMI-barcode filtering strategy. The simplicity and accessibility of this method paves way for widespread use of high-accuracy amplicon sequencing in a variety of genomic applications.

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Section: Resultsmentioning

confidence: 99%

Section: Figure S6: Validation Of Chimera Detectionmentioning

confidence: 99%

Enabling high-accuracy long-read amplicon sequences using unique molecular identifiers with Nanopore or PacBio sequencing

Karst

Ziels

Kirkegaard

et al. 2019

Preprint

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“…PacBio has lower throughput and higher error rates than Illumina but can produce reads that are over 20 kb long at a fraction of the cost of Sanger sequencing. In the last few years, PacBio sequencing has started to be applied to metabarcoding studies, primarily on prokaryotic 16S rDNA (Mosher et al, 2014;Schloss, Jenior, Koumpouras, Westcott, & Highlander, 2016;Wagner et al, 2016) and most recently on larger amplicons also including the 23S rDNA (Martijn et al, 2019). For eukaryotes, the 18S rDNA was nearly fully sequenced for targeted microbial groups (Orr et al, 2018), whilst longer regions also spanning the ITS and the large subunit (LSU) 28S gene were used to analyse fungal diversity (Heeger et al, 2018;Tedersoo & Anslan, 2019;Tedersoo, Tooming-Klunderud, & Anslan, 2018).…”

Section: Introductionmentioning

confidence: 99%

Long‐read metabarcoding of the eukaryotic rDNA operon to phylogenetically and taxonomically resolve environmental diversity

Jamy

Foster

Barbera

et al. 2019

Molecular Ecology Resources

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High‐throughput DNA metabarcoding of amplicon sizes below 500 bp has revolutionized the analysis of environmental microbial diversity. However, these short regions contain limited phylogenetic signal, which makes it impractical to use environmental DNA in full phylogenetic inferences. This lesser phylogenetic resolution of short amplicons may be overcome by new long‐read sequencing technologies. To test this idea, we amplified soil DNA and used PacBio Circular Consensus Sequencing (CCS) to obtain an ~4500‐bp region spanning most of the eukaryotic small subunit (18S) and large subunit (28S) ribosomal DNA genes. We first treated the CCS reads with a novel curation workflow, generating 650 high‐quality operational taxonomic units (OTUs) containing the physically linked 18S and 28S regions. To assign taxonomy to these OTUs, we developed a phylogeny‐aware approach based on the 18S region that showed greater accuracy and sensitivity than similarity‐based methods. The taxonomically annotated OTUs were then combined with available 18S and 28S reference sequences to infer a well‐resolved phylogeny spanning all major groups of eukaryotes, allowing us to accurately derive the evolutionary origin of environmental diversity. A total of 1,019 sequences were included, of which a majority (58%) corresponded to the new long environmental OTUs. The long reads also allowed us to directly investigate the relationships among environmental sequences themselves, which represents a key advantage over the placement of short reads on a reference phylogeny. Together, our results show that long amplicons can be treated in a full phylogenetic framework to provide greater taxonomic resolution and a robust evolutionary perspective to environmental DNA.

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“…Furthermore, the methods used in this study can be applied to any other food-related industry to universally promote our knowledge about microbial transfer during food processing. Continuing advances in long-read sequencing technologies like the release of the Sequel II system and the development of full rRNA operon sequencing strategies will further increase the throughput and taxonomic resolution, offering great potential to implement them in monitoring systems (Martijn et al 2019).…”

Section: Resultsmentioning

confidence: 99%

The sources and transmission routes of microbial populations throughout a meat processing facility

Zwirzitz

Wetzels

Dixon

et al. 2019

Preprint

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Microbial food spoilage is responsible for a considerable amount of waste and can cause food-borne diseases in humans, particularly in immunocompromised individuals and children. Therefore, preventing microbial food spoilage is a major concern for health authorities, regulators, consumers, and the food industry.However, the contamination of food products is difficult to control because there are several potential sources during production, processing, storage, distribution, and consumption, where microorganisms come in contact with the product. Here, we conduct the first study that uses high-throughput full-length 16S rRNA gene sequencing to provide novel insights into bacterial community structure throughout a pork processing plant. Specifically, we investigated what proportion of bacteria on meat are not animal-associated and are therefore transferred during cutting via personnel, equipment, machines, or the slaughter environment. We then created a facility-specific transmission map of bacterial flow which revealed previously unknown sources of bacterial contamination. This allowed us to pinpoint specific taxa to particular environmental sources and provide the facility with essential information for targeted disinfection. For example, Moraxella spp., a prominent meat spoilage organism which was one of the most abundant amplicon sequence variants (ASVs) detected on the meat, was most likely transferred from the gloves of employees, a railing at the classification step, and the polishing tunnel whips. Finally, we provide evidence that 1000 sequences per sample provides a reasonable sequencing depth for microbial source tracking in food processing, suggesting that this approach could be implemented in regular monitoring systems. BackgroundAs the world population is expected to rise to 9.8 billion by 2050, the global demand for food will increase by approximately 70 % in order to satisfy human needs. Resolving this issue, while also reducing greenhouse gas emissions and protecting valuable ecosystems is one of the greatest challenges of our era. Food security experts estimate that 46 % of the required additional food demand can be achieved by increasing food production, whereas the remaining proportion needs to be attained through sustaining the productive capacity (34 %) and better food demand management (20 %) (Keating et al.

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Confident phylogenetic identification of uncultured prokaryotes through long read amplicon sequencing of the 16S‐ITS‐23S rRNA operon

Cited by 59 publications

References 53 publications

Enabling high-accuracy long-read amplicon sequences using unique molecular identifiers with Nanopore or PacBio sequencing

Enabling high-accuracy long-read amplicon sequences using unique molecular identifiers with Nanopore or PacBio sequencing

Long‐read metabarcoding of the eukaryotic rDNA operon to phylogenetically and taxonomically resolve environmental diversity

The sources and transmission routes of microbial populations throughout a meat processing facility

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