Optimised multiplex amplicon sequencing for mutation identification using the MinION nanopore sequencer

Objective: Rapid, cost-effective identification of genetic variants in small candiate genomic regions remains a challenge, particularly for less well equipped or lower throughput laboratories. Application of Oxford Nanopore Technologies' MinION sequencer has the potential to fulfil this requirement. We have developed a multiplexing assay which pools PCR amplicons for MinION sequencing to enable sequencing of multiple templates from multiple individuals which could be applied to gene-targeted diagnostics. Methods: A combined strategy of barcoding and sample pooling was developed for simultaneous multiplex MinION sequencing of 100 PCR amplicons, spanning 30 loci in DNA isolated from 82 neurodevelopmental cases and family members. The target regions were chosen for further interegation because a potentially disease-causative variants had been identified in affected individuals by Illumina exome sequencing. The pooled MinION sequences were deconvoluted by aligning to custom references using the guppy aligner software. Results: Our multiplexing approach produced interpretable and expected sequence from 29 of the 30 targeted genetic loci. The sequence variant which was not correctly resolved in the MinION sequence was adjacent to a five nucleotide homopolymer. It is already known that homopolymers present a resolution problem with the MinION approach. Interstingly despite equimolar quantities of PCR amplicon pooled for sequencing, significant variation in the depth of coverage (139x - 21,499x; mean = 9,050, std err = 538.21) was observed. We observed independent relationships between depth of coverage and target length, and depth of coverage and GC content. These relationships demonstrate biases of the MinION sequencer for longer templates and those with lower GC content. Conclusion: We demonstrate an efficient approach for variant discovery or confirmation from short DNA templates using the MinION sequencing device. With less than 140x depth of coverage required for accurate genotyping, the methodology described here allows for rapid highly multiplexed targeted sequencing of large numbers of samples in a minimally equipped laboratory.

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BrADseq for DNA v1

10.17504/protocols.io.bsx9nfr6 ◽

2021 ◽

Author(s):

S. Thomas Kelly ◽

tsuneo.hakoyama not provided ◽

Kie Kumaishi ◽

Haruka Okuda-Yabukami ◽

Sachi Kato ◽

...

Keyword(s):

Genomic Dna ◽

Genomic Sequence ◽

Sequence Data ◽

Gc Content ◽

Handling Time ◽

Cost Effective ◽

Dna Amount ◽

Dna Libraries ◽

Genomic Regions ◽

Next Generation Sequencing Ngs

The amount of input DNA available to prepare next-generation sequencing (NGS) libraries is often limited, which can lead to GC content bias and enrichment of specific genomic regions with currently available protocols. In this study, we used breath capture technology to incorporate sequencing adapters into DNA to develop a novel cost-effective protocol for the preparation of genomic DNA libraries. We performed a benchmarking experiment comparing our protocol with common commercially available kits for genomic DNA library preparation with input DNA amount in the range of 1 to 50 ng. Our protocol can generate high-quality genomic sequence data with a marked improvement in coverage breadth and low GC bias, in contrast to standard protocols. Further, our protocol reduces sample handling time and reagent costs, and requires comparatively fewer enzymatic steps relative to other protocols, making it suitable for a range of genomics applications.

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

10.1101/645903 ◽

2019 ◽

Cited By ~ 25

Author(s):

Søren M. Karst ◽

Ryan M. Ziels ◽

Rasmus H. Kirkegaard ◽

Emil A. Sørensen ◽

Daniel McDonald ◽

...

Keyword(s):

High Throughput ◽

Single Molecule ◽

Amplicon Sequencing ◽

High Accuracy ◽

Pacbio Sequencing ◽

Consensus Sequences ◽

Oxford Nanopore ◽

Long Read ◽

Genomic Regions ◽

Oxford Nanopore Technologies

AbstractHigh-throughput amplicon sequencing of large genomic regions remains challenging for short-read technologies. Here, we report a high-throughput amplicon sequencing approach combining unique molecular identifiers (UMIs) with Oxford Nanopore Technologies or Pacific Biosciences CCS sequencing, yielding high accuracy single-molecule consensus sequences of large genomic regions. Our approach generates amplicon and genomic sequences of >10,000 bp in length with a mean error-rate of 0.0049-0.0006% and chimera rate <0.022%.

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Deciphering Neurodegenerative Diseases Using Long-Read Sequencing

Neurology ◽

10.1212/wnl.0000000000012466 ◽

2021 ◽

pp. 10.1212/WNL.0000000000012466

Author(s):

Yun Su ◽

Liyuan Fan ◽

Changhe Shi ◽

Tai Wang ◽

Huimin Zheng ◽

...

Keyword(s):

Neurodegenerative Diseases ◽

Single Molecule ◽

Direct Detection ◽

Gc Content ◽

Sequencing Technologies ◽

Oxford Nanopore ◽

Long Read ◽

Repeat Expansions ◽

Genomic Regions

Neurodegenerative diseases exhibit chronic progressive lesions in the central and peripheral nervous systems with unclear causes. The search for pathogenic mutations in human neurodegenerative diseases has benefited from massively parallel short-read sequencers. However, genomic regions, including repetitive elements, especially with high/low GC content, are far beyond the capability of conventional approaches. Recently, long-read single-molecule DNA sequencing technologies have emerged and enabled researchers to study genomes, transcriptomes, and metagenomes at unprecedented resolutions. The identification of novel mutations in unresolved neurodegenerative disorders, the characterization of causative repeat expansions, and the direct detection of epigenetic modifications on naive DNA by virtue of long-read sequencers will further expand our understanding of neurodegenerative diseases. In this paper, we review and compare two prevailing long-read sequencing technologies, Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT), and discuss their applications in neurodegenerative diseases.

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SARS-CoV-2 detection status associates with bacterial community composition in patients and the hospital environment

Microbiome ◽

10.1186/s40168-021-01083-0 ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Clarisse Marotz ◽

Pedro Belda-Ferre ◽

Farhana Ali ◽

Promi Das ◽

Shi Huang ◽

...

Keyword(s):

Health Care Providers ◽

Rna Virus ◽

Bacterial Community Composition ◽

Hospital Setting ◽

Amplicon Sequencing ◽

Hospital Environment ◽

Rrna Gene ◽

Sequence Variant ◽

Care Providers ◽

Single Amplicon

Abstract Background SARS-CoV-2 is an RNA virus responsible for the coronavirus disease 2019 (COVID-19) pandemic. Viruses exist in complex microbial environments, and recent studies have revealed both synergistic and antagonistic effects of specific bacterial taxa on viral prevalence and infectivity. We set out to test whether specific bacterial communities predict SARS-CoV-2 occurrence in a hospital setting. Methods We collected 972 samples from hospitalized patients with COVID-19, their health care providers, and hospital surfaces before, during, and after admission. We screened for SARS-CoV-2 using RT-qPCR, characterized microbial communities using 16S rRNA gene amplicon sequencing, and used these bacterial profiles to classify SARS-CoV-2 RNA detection with a random forest model. Results Sixteen percent of surfaces from COVID-19 patient rooms had detectable SARS-CoV-2 RNA, although infectivity was not assessed. The highest prevalence was in floor samples next to patient beds (39%) and directly outside their rooms (29%). Although bed rail samples more closely resembled the patient microbiome compared to floor samples, SARS-CoV-2 RNA was detected less often in bed rail samples (11%). SARS-CoV-2 positive samples had higher bacterial phylogenetic diversity in both human and surface samples and higher biomass in floor samples. 16S microbial community profiles enabled high classifier accuracy for SARS-CoV-2 status in not only nares, but also forehead, stool, and floor samples. Across these distinct microbial profiles, a single amplicon sequence variant from the genus Rothia strongly predicted SARS-CoV-2 presence across sample types, with greater prevalence in positive surface and human samples, even when compared to samples from patients in other intensive care units prior to the COVID-19 pandemic. Conclusions These results contextualize the vast diversity of microbial niches where SARS-CoV-2 RNA is detected and identify specific bacterial taxa that associate with the viral RNA prevalence both in the host and hospital environment.

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K-seq, an affordable, reliable, and open Klenow NGS-based genotyping technology

Plant Methods ◽

10.1186/s13007-021-00733-6 ◽

2021 ◽

Vol 17 (1) ◽

Author(s):

Peio Ziarsolo ◽

Tomas Hasing ◽

Rebeca Hilario ◽

Victor Garcia-Carpintero ◽

Jose Blanca ◽

...

Keyword(s):

Cost Effective ◽

Genetic Distances ◽

Set Design ◽

Special Equipment ◽

Genetic Studies ◽

Set Up ◽

Genomic Regions ◽

Biology Laboratory ◽

Genotyping Technology ◽

Genetic Results

Abstract Background K-seq, a new genotyping methodology based on the amplification of genomic regions using two steps of Klenow amplification with short oligonucleotides, followed by standard PCR and Illumina sequencing, is presented. The protocol was accompanied by software developed to aid with primer set design. Results As the first examples, K-seq in species as diverse as tomato, dog and wheat was developed. K-seq provided genetic distances similar to those based on WGS in dogs. Experiments comparing K-seq and GBS in tomato showed similar genetic results, although K-seq had the advantage of finding more SNPs for the same number of Illumina reads. The technology reproducibility was tested with two independent runs of the tomato samples, and the correlation coefficient of the SNP coverages between samples was 0.8 and the genotype match was above 94%. K-seq also proved to be useful in polyploid species. The wheat samples generated specific markers for all subgenomes, and the SNPs generated from the diploid ancestors were located in the expected subgenome with accuracies greater than 80%. Conclusion K-seq is an open, patent-unencumbered, easy-to-set-up, cost-effective and reliable technology ready to be used by any molecular biology laboratory without special equipment in many genetic studies.

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Metagenome-validated Parallel Amplicon Sequencing and Text Mining-based Annotations for Simultaneous Profiling of Bacteria and Fungi: Vaginal Microbiome and Mycobiota in Healthy Women

10.21203/rs.3.rs-321778/v1 ◽

2021 ◽

Author(s):

Seppo Virtanen ◽

Schahzad Saqib ◽

Tinja Kanerva ◽

Pekka Nieminen ◽

Ilkka Kalliala ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Cost Effective ◽

Amplicon Sequencing ◽

Rrna Gene ◽

Extensive Literature ◽

Metagenomic Sequencing ◽

Text Extraction ◽

Healthy Women ◽

Bacteria And Fungi

Abstract Background: Amplicon sequencing of kingdom-specific tags such as 16S rRNA gene for bacteria and internal transcribed spacer (ITS) region for fungi are widely used for investigating microbial populations. So far most human studies have focused on bacteria while studies on host-associated fungi in health and disease have only recently started to accumulate. To enable cost-effective parallel analysis of bacterial and fungal communities in human and environmental samples, we developed a method where 16S rRNA gene and ITS-1 amplicons were pooled together for a single Illumina MiSeq or HiSeq run and analysed after primer-based segregation. Taxonomic assignments were performed with Blast in combination with an iterative text-extraction based filtration approach, which uses extensive literature records from public databases to select the most probable hits that were further validated by shotgun metagenomic sequencing. Results: Using 50 vaginal samples, we show that the combined run provides comparable results on bacterial composition and diversity to conventional 16S rRNA gene amplicon sequencing. The text-extraction-based taxonomic assignment guided tool provided ecosystem specific annotations that were confirmed by Metagenomic Phylogenetic Analysis (MetaPhlAn). The metagenome analysis revealed distinct functional differences between the bacterial community types while fungi were undetected, despite being identified in all samples based on ITS amplicons. Co-abundance analysis of bacteria and fungi did not show strong between-kingdom correlations within the vaginal ecosystem of healthy women.Conclusion: Combined amplicon sequencing for bacteria and fungi provides a simple and cost-effective method for simultaneous analysis of microbiota and mycobiota within the same samples. Text extraction-based annotation tool facilitates the characterization and interpretation of defined microbial communities from rapidly accumulating sequencing and metadata readily available through public databases.

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Relative and quantitative rhizosphere microbiome profiling result in distinct abundance patterns

10.1101/2021.02.19.431941 ◽

2021 ◽

Author(s):

Hamed Azarbad ◽

Julien Tremblay ◽

Luke D. Bainard ◽

Etienne Yergeau

Keyword(s):

Water Stress ◽

Relative Abundance ◽

Its Region ◽

Cost Effective ◽

Amplicon Sequencing ◽

Rrna Gene ◽

Real Time Quantitative Pcr ◽

Rhizosphere Microbiome ◽

History Of ◽

Almost All

AbstractNext-generation sequencing is recognized as one of the most popular and cost-effective way of characterizing microbiome in multiple samples. However, most of the currently available amplicon sequencing approaches are inherently limited, as they are often presented based on the relative abundance of microbial taxa, which may not fully represent actual microbiome profiles. Here, we combined amplicon sequencing (16S rRNA gene for bacteria and ITS region for fungi) with real-time quantitative PCR (qPCR) to characterize the rhizosphere microbiome of wheat. We show that the increase in relative abundance of major microbial phyla does not necessarily result in an increase in abundance. One striking observation when comparing relative and quantitative abundances was a substantial increase in the abundance of almost all phyla associated with the rhizosphere of plants grown in soil with no history of water stress as compared with the rhizosphere of plants growing in soil with a history of water stress, which was in contradiction with the trends observed in the relative abundance data. Our results suggest that the estimated absolute abundance approach gives a different perspective than the relative abundance approach, providing complementary information that helps to better understand the rhizosphere microbiome.

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Evaluation of the microbial community structure of potable water samples from occupied and unoccupied buildings using16S rRNA amplicon sequencing

10.1101/2020.07.17.209346 ◽

2020 ◽

Author(s):

Kimothy L Smith ◽

Howard A Shuman ◽

Douglas Findeisen

Keyword(s):

Microbial Community ◽

16S Rrna ◽

Water Samples ◽

Ad Hoc ◽

Microbial Community Composition ◽

Amplicon Sequencing ◽

The Other ◽

Water Usage ◽

16S Rrna Amplicon Sequencing ◽

Oxford Nanopore

AbstractWe conducted two studies of water samples from buildings with normal occupancy and water usage compared to water from buildings that were unoccupied with little or no water usage due to the COVID-19 shutdown. Study 1 had 52 water samples obtained ad hoc from buildings in four metropolitan locations in different states in the US and a range of building types. Study 2 had 36 water samples obtained from two buildings in one metropolitan location with matched water sample types. One of the buildings had been continuously occupied, and the other substantially vacant for approximately 3 months. All water samples were analyzed using 16S rRNA amplicon sequencing with a MinION from Oxford Nanopore Technologies. More than 127 genera of bacteria were identified, including genera with members that are known to include more than 50 putative frank and opportunistic pathogens. While specific results varied among sample locations, 16S rRNA amplicon abundance and the diversity of bacteria were higher in water samples from unoccupied buildings than normally occupied buildings as was the abundance of sequenced amplicons of genera known to include pathogenic bacterial members. In both studies Legionella amplicon abundance was relatively small compared to the abundance of the other bacteria in the samples. Indeed, when present, the relative abundance of Legionella amplicons was lower in samples from unoccupied buildings. Legionella did not predominate in any of the water samples and were found, on average, in 9.6% of samples in Study 1 and 8.3% of samples in Study 2.SynopsisComparison of microbial community composition in the plumbing of occupied and unoccupied buildings during the COVID-19 pandemic shutdown.

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NanoHIV: A Bioinformatics Pipeline for Producing Accurate, Near Full-Length HIV Proviral Genomes Sequenced Using the Oxford Nanopore Technology

Cells ◽

10.3390/cells10102577 ◽

2021 ◽

Vol 10 (10) ◽

pp. 2577

Author(s):

Imogen A. Wright ◽

Kayla E. Delaney ◽

Mary Grace K. Katusiime ◽

Johannes C. Botha ◽

Susan Engelbrecht ◽

...

Keyword(s):

Pcr Amplification ◽

Cost Effective ◽

Full Length ◽

Sequencing Error ◽

Consensus Building ◽

Genome Sequences ◽

Bioinformatics Pipeline ◽

Oxford Nanopore ◽

Single Genome ◽

Hiv 1

HIV-1 proviral single-genome sequencing by limiting-dilution polymerase chain reaction (PCR) amplification is important for differentiating the sequence-intact from defective proviruses that persist during antiretroviral therapy (ART). Intact proviruses may rebound if ART is interrupted and are the barrier to an HIV cure. Oxford Nanopore Technologies (ONT) sequencing offers a promising, cost-effective approach to the sequencing of long amplicons such as near full-length HIV-1 proviruses, but the high diversity of HIV-1 and the ONT sequencing error render analysis of the generated data difficult. NanoHIV is a new tool that uses an iterative consensus generation approach to construct accurate, near full-length HIV-1 proviral single-genome sequences from ONT data. To validate the approach, single-genome sequences generated using NanoHIV consensus building were compared to Illumina® consensus building of the same nine single-genome near full-length amplicons and an average agreement of 99.4% was found between the two sequencing approaches.

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Nanopore amplicon sequencing reveals molecular convergence and local adaptation of opsin genes

10.1101/2020.06.13.150334 ◽

2020 ◽

Author(s):

Katherine M. Eaton ◽

Moisés A. Bernal ◽

Nathan J.C. Backenstose ◽

Trevor J. Krabbenhoft

Keyword(s):

Local Adaptation ◽

Environmental Gradients ◽

Parallel Evolution ◽

Amplicon Sequencing ◽

Species Flock ◽

Closely Related Species ◽

Oxford Nanopore ◽

Opsin Genes ◽

Potential Case ◽

Species Specific

AbstractLocal adaptation can drive diversification of closely related species across environmental gradients and promote convergence of distantly related taxa that experience similar conditions. We examined a potential case of adaptation to novel visual environments in a species flock (Great Lakes salmonids, genus Coregonus) using a new amplicon genotyping protocol on the Oxford Nanopore Flongle. Five visual opsin genes were amplified for individuals of C. artedi, C. hoyi, C. kiyi, and C. zenithicus. Comparisons revealed species-specific differences in the coding sequence of rhodopsin (Tyr261Phe substitution), suggesting local adaptation by C. kiyi to the blue-shifted depths of Lake Superior. Parallel evolution and “toggling” at this amino acid residue has occurred several times across the fish tree of life, resulting in identical changes to the visual systems of distantly related taxa across replicated environmental gradients. Our results suggest that ecological differences and local adaptation to distinct visual environments are strong drivers of both evolutionary parallelism and diversification.

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