Investigation of chimeric reads using the MinION

Following a nanopore sequencing run of PCR products of three amplicons less than 1kb, an abundance of reads failed quality control due to template/complement mismatch. A BLAST search demonstrated that some of the failed reads mapped to two different genes -- an unexpected observation, given that PCR was carried out separately for each amplicon. A further investigation was carried out specifically to search for chimeric reads, using separate barcodes for each amplicon and trying two different ligation methods prior to sample loading. Despite the separation of ligation products, chimeric reads formed from different amplicons were still observed in the base-called sequence.The long-read nature of nanopore sequencing presents an effective tool for the discovery and filtering of chimeric reads. We have found that at least 1.7% of reads prepared using the Nanopore LSK002 2D Ligation Kit include post-amplification chimeric elements. This finding has potential implications for other amplicon sequencing technologies, as the process is unlikely to be specific to the sample preparation used for nanopore sequencing.

Download Full-text

Evaluation of Germline Structural Variant Calling Methods for Nanopore Sequencing Data

Frontiers in Genetics ◽

10.3389/fgene.2021.761791 ◽

2021 ◽

Vol 12 ◽

Author(s):

Davide Bolognini ◽

Alberto Magi

Keyword(s):

Variant Calling ◽

Research Report ◽

Nanopore Sequencing ◽

Sequencing Data ◽

Factors Affecting ◽

Sequencing Technologies ◽

Long Reads ◽

Oxford Nanopore ◽

Sequencing Studies ◽

Long Read

Structural variants (SVs) are genomic rearrangements that involve at least 50 nucleotides and are known to have a serious impact on human health. While prior short-read sequencing technologies have often proved inadequate for a comprehensive assessment of structural variation, more recent long reads from Oxford Nanopore Technologies have already been proven invaluable for the discovery of large SVs and hold the potential to facilitate the resolution of the full SV spectrum. With many long-read sequencing studies to follow, it is crucial to assess factors affecting current SV calling pipelines for nanopore sequencing data. In this brief research report, we evaluate and compare the performances of five long-read SV callers across four long-read aligners using both real and synthetic nanopore datasets. In particular, we focus on the effects of read alignment, sequencing coverage, and variant allele depth on the detection and genotyping of SVs of different types and size ranges and provide insights into precision and recall of SV callsets generated by integrating the various long-read aligners and SV callers. The computational pipeline we propose is publicly available at https://github.com/davidebolo1993/EViNCe and can be adjusted to further evaluate future nanopore sequencing datasets.

Download Full-text

Long read nanopore sequencing for detection of HLA and CYP2D6 variants and haplotypes

F1000Research ◽

10.12688/f1000research.6037.2 ◽

2015 ◽

Vol 4 ◽

pp. 17 ◽

Cited By ~ 55

Author(s):

Ron Ammar ◽

Tara A. Paton ◽

Dax Torti ◽

Adam Shlien ◽

Gary D. Bader

Keyword(s):

Medical Decision ◽

Nanopore Sequencing ◽

Clinical Environment ◽

Sequencing Technologies ◽

Long Reads ◽

Long Read ◽

Complete Genomics ◽

Nanopore Sequencer ◽

Actionable Findings ◽

Haplotype Information

Haplotypes are often critical for the interpretation of genetic laboratory observations into medically actionable findings. Current massively parallel DNA sequencing technologies produce short sequence reads that are often unable to resolve haplotype information. Phasing short read data typically requires supplemental statistical phasing based on known haplotype structure in the population or parental genotypic data. Here we demonstrate that the MinION nanopore sequencer is capable of producing very long reads to resolve both variants and haplotypes of HLA-A, HLA-B and CYP2D6 genes important in determining patient drug response in sample NA12878 of CEPH/UTAH pedigree 1463, without the need for statistical phasing. Long read data from a single 24-hour nanopore sequencing run was used to reconstruct haplotypes, which were confirmed by HapMap data and statistically phased Complete Genomics and Sequenom genotypes. Our results demonstrate that nanopore sequencing is an emerging standalone technology with potential utility in a clinical environment to aid in medical decision-making.

Download Full-text

Long read nanopore sequencing for detection of HLA and CYP2D6 variants and haplotypes

F1000Research ◽

10.12688/f1000research.6037.1 ◽

2015 ◽

Vol 4 ◽

pp. 17 ◽

Cited By ~ 8

Author(s):

Ron Ammar ◽

Tara A. Paton ◽

Dax Torti ◽

Adam Shlien ◽

Gary D. Bader

Keyword(s):

Medical Decision ◽

Nanopore Sequencing ◽

Clinical Environment ◽

Sequencing Technologies ◽

Long Reads ◽

Long Read ◽

Complete Genomics ◽

Nanopore Sequencer ◽

Actionable Findings ◽

Haplotype Information

Haplotypes are often critical for the interpretation of genetic laboratory observations into medically actionable findings. Current massively parallel DNA sequencing technologies produce short sequence reads that are often unable to resolve haplotype information. Phasing short read data typically requires supplemental statistical phasing based on known haplotype structure in the population or parental genotypic data. Here we demonstrate that the MinION nanopore sequencer is capable of producing very long reads to resolve both variants and haplotypes of HLA-A, HLA-B and CYP2D6 genes important in determining patient drug response in sample NA12878 of CEPH/UTAH pedigree 1463, without the need for statistical phasing. Long read data from a single 24-hour nanopore sequencing run was used to reconstruct haplotypes, which were confirmed by HapMap data and statistically phased Complete Genomics and Sequenom genotypes. Our results demonstrate that nanopore sequencing is an emerging standalone technology with potential utility in a clinical environment to aid in medical decision-making.

Download Full-text

Perspectives and benefits of high-throughput long-read sequencing in microbial ecology

Applied and Environmental Microbiology ◽

10.1128/aem.00626-21 ◽

2021 ◽

Author(s):

Leho Tedersoo ◽

Mads Albertsen ◽

Sten Anslan ◽

Benjamin Callahan

Keyword(s):

Microbial Ecology ◽

High Throughput ◽

Single Molecule ◽

High Throughput Sequencing ◽

Environmental Dna ◽

Nanopore Sequencing ◽

High Quality ◽

Short Read ◽

Sequencing Technologies ◽

Long Read

Short-read, high-throughput sequencing (HTS) methods have yielded numerous important insights into microbial ecology and function. Yet, in many instances short-read HTS techniques are suboptimal, for example by providing insufficient phylogenetic resolution or low integrity of assembled genomes. Single-molecule and synthetic long-read (SLR) HTS methods have successfully ameliorated these limitations. In addition, nanopore sequencing has generated a number of unique analysis opportunities such as rapid molecular diagnostics and direct RNA sequencing, and both PacBio and nanopore sequencing support detection of epigenetic modifications. Although initially suffering from relatively low sequence quality, recent advances have greatly improved the accuracy of long read sequencing technologies. In spite of great technological progress in recent years, the long-read HTS methods (PacBio and nanopore sequencing) are still relatively costly, require large amounts of high-quality starting material, and commonly need specific solutions in various analysis steps. Despite these challenges, long-read sequencing technologies offer high-quality, cutting-edge alternatives for testing hypotheses about microbiome structure and functioning as well as assembly of eukaryote genomes from complex environmental DNA samples.

Download Full-text

Long-read amplicon denoising

10.1101/383794 ◽

2018 ◽

Cited By ~ 3

Author(s):

Venkatesh Kumar ◽

Thomas Vollbrecht ◽

Mark Chernyshev ◽

Sanjay Mohan ◽

Brian Hanst ◽

...

Keyword(s):

Ground Truth ◽

Amplicon Sequencing ◽

Error Rates ◽

Sequencing Error ◽

Short Read Sequencing ◽

Sequencing Technologies ◽

Medium Length ◽

Long Reads ◽

Long Read ◽

Error Profiles

Long-read next generation amplicon sequencing shows promise for studying complete genes or genomes from complex and diverse populations. Current long-read sequencing technologies have challenging error profiles, hindering data processing and incorporation into downstream analyses. Here we consider the problem of how to reconstruct, free of sequencing error, the true sequence variants and their associated frequencies. Called “amplicon denoising”, this problem has been extensively studied for short-read sequencing technologies, but current solutions do not appear to generalize well to long reads with high indel error rates. We introduce two methods: one that runs nearly instantly and is very accurate for medium length reads (here ~2.6kb) and high template coverage, and another, slower method that is more robust when reads are very long or coverage is lower.On one real dataset with ground truth, and on a number of simulated datasets, we compare our two approaches to each other and to existing algorithms. We outperform all tested methods in accuracy, with competitive run times even for our slower method.Fast Amplicon Denoising (FAD) and Robust Amplicon Denoising (RAD) are implemented purely in the Julia scientific computing language, and are hereby released along with a complete toolkit of functions that allow long-read amplicon sequence analysis pipelines to be constructed in pure Julia. Further, we make available a webserver to dramatically simplify the processing of long-read PacBio sequences.

Download Full-text

Beginner’s guide to next-generation sequencing

The Biochemist ◽

10.1042/bio_2021_135 ◽

2021 ◽

Author(s):

Louise Aigrain

Keyword(s):

Next Generation Sequencing ◽

Sample Preparation ◽

Third Generation ◽

Next Generation ◽

Sequencing Technologies ◽

Long Read ◽

Pros And Cons ◽

The Cost ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

Since the publication of the first draft of the human genome 20 years ago, several novel sequencing technologies have emerged. Whilst some drive the cost of DNA sequencing down, others address the difficult parts of the genome which remained inaccessible so far. But the next-generation sequencing (NGS) landscape is a fast-changing environment and one can easily get lost between second- and third- generation sequencers, or the pros and cons of short- versus long-read technologies. In this beginner’s guide to NGS, we will review the main NGS technologies available in 2021. We will compare sample preparation protocols and sequencing methods, highlighting the requirements and advantages of each technology.

Download Full-text

De novo whole-genome assembly of a wild type yeast isolate using nanopore sequencing

F1000Research ◽

10.12688/f1000research.11146.2 ◽

2018 ◽

Vol 6 ◽

pp. 618 ◽

Cited By ~ 2

Author(s):

Michael Liem ◽

Hans J. Jansen ◽

Ron P. Dirks ◽

Christiaan V. Henkel ◽

G. Paul H. van Heusden ◽

...

Keyword(s):

De Novo ◽

Sequence Data ◽

New Technology ◽

Whole Genome ◽

Nanopore Sequencing ◽

Short Read ◽

Sequencing Technologies ◽

Oxford Nanopore ◽

Before And After ◽

Long Read

Background: The introduction of the MinION sequencing device by Oxford Nanopore Technologies may greatly accelerate whole genome sequencing. Nanopore sequence data offers great potential for de novo assembly of complex genomes without using other technologies. Furthermore, Nanopore data combined with other sequencing technologies is highly useful for accurate annotation of all genes in the genome. In this manuscript we used nanopore sequencing as a tool to classify yeast strains. Methods: We compared various technical and software developments for the nanopore sequencing protocol, showing that the R9 chemistry is, as predicted, higher in quality than R7.3 chemistry. The R9 chemistry is an essential improvement for assembly of the extremely AT-rich mitochondrial genome. We double corrected assemblies from four different assemblers with PILON and assessed sequence correctness before and after PILON correction with a set of 290 Fungi genes using BUSCO. Results: In this study, we used this new technology to sequence and de novo assemble the genome of a recently isolated ethanologenic yeast strain, and compared the results with those obtained by classical Illumina short read sequencing. This strain was originally named Candida vartiovaarae (Torulopsis vartiovaarae) based on ribosomal RNA sequencing. We show that the assembly using nanopore data is much more contiguous than the assembly using short read data. We also compared various technical and software developments for the nanopore sequencing protocol, showing that nanopore-derived assemblies provide the highest contiguity. Conclusions: The mitochondrial and chromosomal genome sequences showed that our strain is clearly distinct from other yeast taxons and most closely related to published Cyberlindnera species. In conclusion, MinION-mediated long read sequencing can be used for high quality de novo assembly of new eukaryotic microbial genomes.

Download Full-text

Amplicon Sequencing in the Era of Highly-Accurate Long Reads

ARPHA Conference Abstracts ◽

10.3897/aca.4.e65405 ◽

2021 ◽

Vol 4 ◽

Author(s):

Benjamin Callahan

Keyword(s):

Dna Sequencing ◽

New Technology ◽

Amplicon Sequencing ◽

Error Rates ◽

Important Advance ◽

Sequencing Technologies ◽

Long Reads ◽

Long Read

An important advance in DNA sequencing has been the development of long-read sequencing technologies that produce sequencing reads of tens to hundreds of kilobases in length. However, these technologies typically have high (~8%) per-base error rates. Recently, an effectively new technology I call highly-accurate long-read sequencing has been developed, that allows for the generation of multi-kilobase reads with extremely high per-base accuracies (>99.9%). I will present and evaluate two such technologies, PacBio HiFi and LoopSeq SLR sequencing, and discuss potential metabarcoding applications of highly-accurate long-read amplicon sequencing in general.

Download Full-text

De novo whole-genome assembly of a wild type yeast isolate using nanopore sequencing

F1000Research ◽

10.12688/f1000research.11146.1 ◽

2017 ◽

Vol 6 ◽

pp. 618 ◽

Cited By ~ 3

Author(s):

Hans J. Jansen ◽

Ron P. Dirks ◽

Michael Liem ◽

Christiaan V. Henkel ◽

G. Paul H. van Heusden ◽

...

Keyword(s):

De Novo ◽

Sequence Data ◽

New Technology ◽

Whole Genome ◽

Nanopore Sequencing ◽

Short Read ◽

Sequencing Technologies ◽

Oxford Nanopore ◽

Long Read ◽

Wild Type Yeast

Background: The introduction of the MinIONTM sequencing device by Oxford Nanopore Technologies may greatly accelerate whole genome sequencing. It has been shown that the nanopore sequence data, in combination with other sequencing technologies, is highly useful for accurate annotation of all genes in the genome. However, it also offers great potential for de novo assembly of complex genomes without using other technologies. In this manuscript we used nanopore sequencing as a tool to classify yeast strains. Methods: We compared various technical and software developments for the nanopore sequencing protocol, showing that the R9 chemistry is, as predicted, higher in quality than R7.3 chemistry. The R9 chemistry is an essential improvement for assembly of the extremely AT-rich mitochondrial genome. Results: In this study, we used this new technology to sequence and de novo assemble the genome of a recently isolated ethanologenic yeast strain, and compared the results with those obtained by classical Illumina short read sequencing. This strain was originally named Candida vartiovaarae (Torulopsis vartiovaarae) based on ribosomal RNA sequencing. We show that the assembly using nanopore data is much more contiguous than the assembly using short read data. Conclusions: The mitochondrial and chromosomal genome sequences showed that our strain is clearly distinct from other yeast taxons and most closely related to published Cyberlindnera species. In conclusion, MinION-mediated long read sequencing can be used for high quality de novo assembly of new eukaryotic microbial genomes.

Download Full-text