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

Karst, Søren Michael; Ziels, Ryan M.; Kirkegaard, Rasmus Hansen; Sørensen, Emil A.; McDonald, Daniel; Zhu, Quanyin; Knight, Rob; Albertsen, Mads

doi:10.1038/s41592-020-01041-y

Cited by 250 publications

(287 citation statements)

References 55 publications

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“…Accuracy is essential to many amplicon sequencing applications, and the level of accuracy achieved by Loop-Seq may open up new opportunities. Long-read amplicon sequencing using the PacBio and Oxford Nanopore technologies ( [12]; Eren 2019 [24];) has received increased recent attention, with encouraging results demonstrating that per-base accuracy exceeding common short-read approaches can be obtained by combining long-read sequencing with molecular methods such as the construction of PacBio circular consensus (CCS) reads and appropriate bioinformatics. In the bacterial profiling application, multiple studies have shown that substantial improvements in species and subspecies resolution can be achieved by sequencing the entire~1.5 kb 16S gene, rather than just segments of 100-500 bases as is most commonly practiced today, and that even greater resolution is achievable by extending the sequenced region to most or all of the rrn operon [30].…”

Section: Discussionmentioning

confidence: 99%

Ultra-accurate microbial amplicon sequencing with synthetic long reads

et al. 2021

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Background Out of the many pathogenic bacterial species that are known, only a fraction are readily identifiable directly from a complex microbial community using standard next generation DNA sequencing. Long-read sequencing offers the potential to identify a wider range of species and to differentiate between strains within a species, but attaining sufficient accuracy in complex metagenomes remains a challenge. Methods Here, we describe and analytically validate LoopSeq, a commercially available synthetic long-read (SLR) sequencing technology that generates highly accurate long reads from standard short reads. Results LoopSeq reads are sufficiently long and accurate to identify microbial genes and species directly from complex samples. LoopSeq perfectly recovered the full diversity of 16S rRNA genes from known strains in a synthetic microbial community. Full-length LoopSeq reads had a per-base error rate of 0.005%, which exceeds the accuracy reported for other long-read sequencing technologies. 18S-ITS and genomic sequencing of fungal and bacterial isolates confirmed that LoopSeq sequencing maintains that accuracy for reads up to 6 kb in length. LoopSeq full-length 16S rRNA reads could accurately classify organisms down to the species level in rinsate from retail meat samples, and could differentiate strains within species identified by the CDC as potential foodborne pathogens. Conclusions The order-of-magnitude improvement in length and accuracy over standard Illumina amplicon sequencing achieved with LoopSeq enables accurate species-level and strain identification from complex- to low-biomass microbiome samples. The ability to generate accurate and long microbiome sequencing reads using standard short read sequencers will accelerate the building of quality microbial sequence databases and removes a significant hurdle on the path to precision microbial genomics.

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Section: Discussionmentioning

confidence: 99%

Ultra-accurate microbial amplicon sequencing with synthetic long reads

et al. 2021

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“…Our findings warrant larger studies in these and other dengue surveillance cohorts, as well as exploration of how this approach can be used for other arboviruses such as chikungunya and Zika viruses. Ideally, such studies should be coupled with advances in the accuracy of long-read deployable sequencing platforms that could permit near real-time intra-host variant construction ( 46 ).…”

Section: Discussionmentioning

confidence: 99%

Precision Tracing of Household Dengue Spread Using Inter- and Intra-Host Viral Variation Data, Kamphaeng Phet, Thailand

Berry¹,

Melendrez²,

Pollett³

et al. 2021

Emerg. Infect. Dis.

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D engue virus (DENV) causes an estimated 390 million infections each year, 96 million of which manifest as clinical disease (1). Dengue is endemic in >100 countries. An estimated 3.9 billion persons are at risk for infection, of which 75% reside in the Asia-Pacifi c region (2). DENV control approaches are best informed by granular spatial epidemiology of these viruses, and investigators have long tried to understand the landscape of DENV dynamics and spread. Robust public health surveillance and rigorous academic research have enabled tracking of clinical infections to understand changing demographics, populations at risk, hyperendemicity, transmission, disease severity, and virus dispersal patterns within and across human populations (3-10). With the advent of sequencing technologies, single-gene and whole-genome DENV analyses have complemented case-based and serologic surveillance and enabled further insights into DENV epidemic dynamics and spread, disease severity, introductions and emergence of novel variants, tracking of DENV transmissions, and monitoring of viral diversity and evolution (11-17). Investigations of DENV infection dynamics, from the scales of countries and districts down to the city, village, school, and house level, have been performed to describe the patterns and predictors of DENV spread (7,9,(11)(12)(13)(18)(19)(20)(21). These studies have enabled many novel insights into DENV transmission and contributed to improved prediction, prevention, and control strategies. Such studies have also emphasized a key limitation of DENV genomic epidemiology: reconstructing transmission chains between households and within households is typically not possible, even though resolving DENV spread at such fi ne scales would offer major relevance to public health response.Consensus whole-genome sequences, which are typically used in genomic studies of viral epidemics and spread, typically have insuffi cient variability to distinguish between infecting strains sampled within 2 weeks of each other, especially if closely sampled in space ( 22). This limitation might lead to unresolved

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“…While its long-read length (of up to ∼1 Mb, [6]) makes it very attractive for the generation of de novo genome assemblies (see e.g. [6,7]), its low upfront costs (∼1000 USD) and its portability offer huge advantages for DNA-or metabarcoding experiments in countries with limited infrastructure and funds for molecular biomonitoring [8][9][10][11][12], and for teaching and local capacity building [13,14]. The use of the MinION has been extensively investigated for biodiversity research and biomonitoring projects (reviewed in [15]).…”

Section: Applications To Species Identificationmentioning

confidence: 99%

Nanopore sequencing in non-human forensic genetics

Ogden

Vasiljevic

Prost

2021

Emerging Topics in Life Sciences

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The past decade has seen a rapid expansion of non-human forensic genetics coinciding with the development of 2nd and 3rd generation DNA sequencing technologies. Nanopore sequencing is one such technology that offers massively parallel sequencing at a fraction of the capital cost of other sequencing platforms. The application of nanopore sequencing to species identification has already been widely demonstrated in biomonitoring studies and has significant potential for non-human forensic casework, particularly in the area of wildlife forensics. This review examines nanopore sequencing technology and assesses its potential applications, advantages and drawbacks for use in non-human forensics, alongside other next-generation sequencing platforms and as a possible replacement to Sanger sequencing. We assess the specific challenges of sequence error rate and the standardisation of consensus sequence production, before discussing recent progress in the validation of nanopore sequencing for use in forensic casework. We conclude that nanopore sequencing may be able to play a considerable role in the future of non-human forensic genetics, especially for applications to wildlife law enforcement within emerging forensic laboratories.

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

Cited by 250 publications

References 55 publications

Ultra-accurate microbial amplicon sequencing with synthetic long reads

Ultra-accurate microbial amplicon sequencing with synthetic long reads

Precision Tracing of Household Dengue Spread Using Inter- and Intra-Host Viral Variation Data, Kamphaeng Phet, Thailand

Nanopore sequencing in non-human forensic genetics

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