Re-examination of two diatom reference genomes using long-read sequencing

Abstract Background The marine diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum are valuable model organisms for exploring the evolution, diversity and ecology of this important algal group. Their reference genomes, published in 2004 and 2008, respectively, were the product of traditional Sanger sequencing. In the case of T. pseudonana, optical restriction site mapping was employed to further clarify and contextualize chromosome-level scaffolds. While both genomes are considered highly accurate and reasonably contiguous, they still contain many unresolved regions and unordered/unlinked scaffolds. Results We have used Oxford Nanopore Technologies long-read sequencing to update and validate the quality and contiguity of the T. pseudonana and P. tricornutum genomes. Fine-scale assessment of our long-read derived genome assemblies allowed us to resolve previously uncertain genomic regions, further characterize complex structural variation, and re-evaluate the repetitive DNA content of both genomes. We also identified 1862 previously undescribed genes in T. pseudonana. In P. tricornutum, we used transposable element detection software to identify 33 novel copia-type LTR-RT insertions, indicating ongoing activity and rapid expansion of this superfamily as the organism continues to be maintained in culture. Finally, Bionano optical mapping of P. tricornutum chromosomes was combined with long-read sequence data to explore the potential of long-read sequencing and optical mapping for resolving haplotypes. Conclusion Despite its potential to yield highly contiguous scaffolds, long-read sequencing is not a panacea. Even for relatively small nuclear genomes such as those investigated herein, repetitive DNA sequences cause problems for current genome assembly algorithms. Determining whether a long-read derived genomic assembly is ‘better’ than one produced using traditional sequence data is not straightforward. Our revised reference genomes for P. tricornutum and T. pseudonana nevertheless provide additional insight into the structure and evolution of both genomes, thereby providing a more robust foundation for future diatom research.

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Long-read, whole-genome shotgun sequence data for five model organisms

Scientific Data ◽

10.1038/sdata.2014.45 ◽

2014 ◽

Vol 1 (1) ◽

Cited By ~ 89

Author(s):

Kristi E Kim ◽

Paul Peluso ◽

Primo Babayan ◽

P. Jane Yeadon ◽

Charles Yu ◽

...

Keyword(s):

Sequence Data ◽

Whole Genome Shotgun ◽

Model Organisms ◽

Whole Genome ◽

Shotgun Sequence ◽

Whole Genome Shotgun Sequence ◽

Long Read ◽

Genome Shotgun Sequence

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BleTIES: Annotation of natural genome editing in ciliates using long read sequencing

10.1101/2021.05.18.444610 ◽

2021 ◽

Author(s):

Brandon K. B. Seah ◽

Estienne C. Swart

Keyword(s):

Dna Sequences ◽

Sequence Data ◽

Low Complexity ◽

Supplementary Information ◽

Neighboring Element ◽

Sequencing Technologies ◽

Long Reads ◽

Oxford Nanopore ◽

Long Read ◽

Element Elimination

Ciliates are single-celled eukaryotes that eliminate specific, interspersed DNA sequences (internally eliminated sequences, IESs) from their genomes during development. These are challenging to annotate and assemble because IES-containing sequences are much less abundant in the cell than those without, and IES sequences themselves often contain repetitive and low-complexity sequences. Long read sequencing technologies from Pacific Biosciences and Oxford Nanopore have the potential to reconstruct longer IESs than has been possible with short reads, and also the ability to detect correlations of neighboring element elimination. Here we present BleTIES, a software toolkit for detecting, assembling, and analyzing IESs using mapped long reads. Availability and implementation: BleTIES is implemented in Python 3. Source code is available at https://github.com/Swart-lab/bleties (MIT license), and also distributed via Bioconda. Contact: [email protected] Supplementary information: Benchmarking of BleTIES with published sequence data.

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WeFaceNano: a user-friendly pipeline for complete ONT sequence assembly and detection of antibiotic resistance in multi-plasmid bacterial isolates

BMC Microbiology ◽

10.1186/s12866-021-02225-y ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Astrid P. Heikema ◽

Rick Jansen ◽

Saskia D. Hiltemann ◽

John P. Hays ◽

Andrew P. Stubbs

Keyword(s):

Antibiotic Resistance ◽

Resistance Genes ◽

Dna Sequences ◽

Sequence Data ◽

Antibiotic Resistance Genes ◽

Clinical Samples ◽

Microbial Resistance ◽

Sequencing Platform ◽

Long Read ◽

User Friendly

Abstract Background Bacterial plasmids often carry antibiotic resistance genes and are a significant factor in the spread of antibiotic resistance. The ability to completely assemble plasmid sequences would facilitate the localization of antibiotic resistance genes, the identification of genes that promote plasmid transmission and the accurate tracking of plasmid mobility. However, the complete assembly of plasmid sequences using the currently most widely used sequencing platform (Illumina-based sequencing) is restricted due to the generation of short sequence lengths. The long-read Oxford Nanopore Technologies (ONT) sequencing platform overcomes this limitation. Still, the assembly of plasmid sequence data remains challenging due to software incompatibility with long-reads and the error rate generated using ONT sequencing. Bioinformatics pipelines have been developed for ONT-generated sequencing but require computational skills that frequently are beyond the abilities of scientific researchers. To overcome this challenge, the authors developed ‘WeFaceNano’, a user-friendly Web interFace for rapid assembly and analysis of plasmid DNA sequences generated using the ONT platform. WeFaceNano includes: a read statistics report; two assemblers (Miniasm and Flye); BLAST searching; the detection of antibiotic resistance- and replicon genes and several plasmid visualizations. A user-friendly interface displays the main features of WeFaceNano and gives access to the analysis tools. Results Publicly available ONT sequence data of 21 plasmids were used to validate WeFaceNano, with plasmid assemblages and anti-microbial resistance gene detection being concordant with the published results. Interestingly, the “Flye” assembler with “meta” settings generated the most complete plasmids. Conclusions WeFaceNano is a user-friendly open-source software pipeline suitable for accurate plasmid assembly and the detection of anti-microbial resistance genes in (clinical) samples where multiple plasmids can be present.

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Accurate sequence variant genotyping in cattle using variation-aware genome graphs

10.1101/460345 ◽

2018 ◽

Cited By ~ 1

Author(s):

Danang Crysnanto ◽

Christine Wurmser ◽

Hubert Pausch

Keyword(s):

Dna Sequences ◽

Reference Genome ◽

Sequence Data ◽

Sequence Variant ◽

Sequence Variants ◽

Sequencing Data ◽

Reference Allele ◽

Reference Genomes ◽

Genome Graph ◽

Genotype Concordance

Background: The genotyping of sequence variants typically involves as a first step the alignment of sequencing reads to a linear reference genome. Because a linear reference genome represents only a small fraction of sequence variation within a species, reference allele bias may occur at highly polymorphic or diverged regions of the genome. Graph-based methods facilitate to compare sequencing reads to a variation-aware genome graph that incorporates non-redundant DNA sequences that segregate within a species. We compared accuracy and sensitivity of graph-based sequence variant genotyping using the Graphtyper software to two widely used methods, i.e., GATK and SAMtools, that rely on linear reference genomes using whole-genomes sequencing data of 49 Original Braunvieh cattle. Results: We discovered 21,140,196, 20,262,913 and 20,668,459 polymorphic sites using GATK, Graphtyper, and SAMtools, respectively. Comparisons between sequence variant and microarray-derived genotypes showed that Graphtyper outperformed both GATK and SAMtools in terms of genotype concordance, non-reference sensitivity, and non-reference discrepancy. The sequence variant genotypes that were obtained using Graphtyper had the lowest number of mendelian inconsistencies for both SNPs and indels in nine sire-son pairs with sequence data. Genotype phasing and imputation using the Beagle software improved the quality of the sequence variant genotypes for all tools evaluated particularly for animals that have been sequenced at low coverage. Following imputation, the concordance between sequence- and microarray-derived genotypes was almost identical for the three methods evaluated, i.e., 99.32, 99.46, and 99.24 % for GATK, Graphtyper, and SAMtools, respectively. Variant filtration based on commonly used criteria improved the genotype concordance slightly but it also decreased sensitivity. Graphtyper required considerably more computing resources than SAMtools but it required less than GATK. Conclusions: Sequence variant genotyping using Graphtyper is accurate, sensitive and computationally feasible in cattle. Graph-based methods enable sequence variant genotyping from variation-aware reference genomes that may incorporate cohort-specific sequence variants which is not possible with the current implementations of state-of-the-art methods that rely on linear reference genomes.

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Using MinION nanopore sequencing to generate a de novo eukaryotic draft genome: preliminary physiological and genomic description of the extremophilic red alga Galdieria sulphuraria strain SAG 107.79

10.1101/076208 ◽

2016 ◽

Cited By ~ 5

Author(s):

Amanda M. Davis ◽

Manuela Iovinella ◽

Sally James ◽

Thomas Robshaw ◽

Jennifer R. Dodson ◽

...

Keyword(s):

Dna Sequences ◽

Genome Assembly ◽

De Novo ◽

Sequence Data ◽

Draft Genome ◽

Carbon Sources ◽

Red Alga ◽

Full Genome Sequencing ◽

Galdieria Sulphuraria ◽

Long Read

AbstractWe report here the de novo assembly of a eukaryotic genome using only MinION nanopore DNA sequence data by examining a novel Galdieria sulphuraria genome: strain SAG 107.79. This extremophilic red alga was targeted for full genome sequencing as we found that it could grow on a wide variety of carbon sources and could uptake several precious and rare-earth metals, which places it as an interesting biological target for disparate industrial biotechnological uses. Phylogenetic analysis clearly places this as a species of G. sulphuraria. Here we additionally show that the genome assembly generated via nanopore long read data was of a high quality with regards to low total number of contiguous DNA sequences and long length of assemblies. Collectively, the MinION platform looks to rival other competing approaches for de novo genome acquisition with available informatics tools for assembly. The genome assembly is publically released as NCBI BioProject PRJNA330791. Further work is needed to reduce small insertion-deletion errors, relative to short-read assemblies.

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Quantitative and qualitative genomic characterization of cultivated Ilex L. species

Plant Genetic Resources ◽

10.1017/s1479262114000756 ◽

2014 ◽

Vol 13 (2) ◽

pp. 142-152 ◽

Cited By ~ 4

Author(s):

Alexandra Marina Gottlieb ◽

Lidia Poggio

Keyword(s):

Genome Size ◽

Repetitive Dna ◽

Dna Sequences ◽

Sequence Data ◽

Repetitive Sequences ◽

Representational Difference Analysis ◽

Ilex Paraguariensis ◽

Repetitive Dna Sequences ◽

A Genome

The development of modern approaches to the genetic improvement of the tree crops Ilex paraguariensis (‘yerba mate’) and Ilex dumosa (‘yerba señorita’) is halted by the scarcity of basic genetic information. In this study, we characterized the implementation of low-cost methodologies such as representational difference analysis (RDA), single-strand conformation polymorphisms (SSCP), and reverse and direct dot-blot filter hybridization assays coupled with thorough bioinformatic characterization of sequence data for both species. Also, we estimated the genome size of each species using flow cytometry. This study contributes to the better understanding of the genetic differences between two cultivated species, by generating new quantitative and qualitative genome-level data. Using the RDA technique, we isolated a group of non-coding repetitive sequences, tentatively considered as Ilex-specific, which were 1.21- to 39.62-fold more abundant in the genome of I. paraguariensis. Another group of repetitive DNA sequences involved retrotransposons, which appeared 1.41- to 35.77-fold more abundantly in the genome of I. dumosa. The genomic DNA of each species showed different performances in filter hybridizations: while I. paraguariensis showed a high intraspecific affinity, I. dumosa exhibited a higher affinity for the genome of the former species (i.e. interspecific). These differences could be attributed to the occurrence of homologous but slightly divergent repetitive DNA sequences, highly amplified in the genome of I. paraguariensis but not in the genome of I. dumosa. Additionally, our hybridization outcomes suggest that the genomes of both species have less than 80% similarity. Moreover, for the first time, we report herein a genome size estimate of 1670 Mbp for I. paraguariensis and that of 1848 Mbp for I. dumosa.

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RepeatProfiler: a pipeline for visualization and comparative analysis of repetitive DNA profiles

10.1101/2020.05.22.111252 ◽

2020 ◽

Cited By ~ 1

Author(s):

S. Negm ◽

A. Greenberg ◽

A.M. Larracuente ◽

J.S. Sproul

Keyword(s):

Repetitive Dna ◽

Sequence Data ◽

Repetitive Sequences ◽

Single Copy ◽

Model Organisms ◽

Phenotypic Change ◽

Dna Repeats ◽

High Resolution Data ◽

Genomic Resources ◽

Dna Profiles

AbstractStudy of DNA repeats in model organisms highlights the role of repetitive DNA in many processes that drive genome evolution and phenotypic change. Because repetitive DNA is much more dynamic than single-copy DNA, repetitive sequences can reveal signals of evolutionary history over short time scales that may not be evident in sequences from slower-evolving genomic regions. Many tools for studying repeats are directed toward organisms with existing genomic resources, including genome assemblies and repeat libraries. However, signals in repeat variation may prove especially valuable in disentangling evolutionary histories in diverse non-model groups, for which genomic resources are limited. Here we introduce RepeatProfiler, a tool for generating, visualizing, and comparing repetitive DNA profiles from low-coverage, short-read sequence data. RepeatProfiler automates the generation and visualization of repetitive DNA coverage depth profiles and allows for statistical comparison of profile shape across samples. In addition, RepeatProfiler facilitates comparison of profiles by extracting signal from sequence variants across profiles which can then be analyzed as molecular morphological characters using phylogenetic analysis. We validate RepeatProfiler with data sets from ground beetles (Bembidion), flies (Drosophila), and tomatoes (Solanum). We highlight the potential of repetitive DNA profiles as a high-resolution data source for studies in species delimitation, comparative genomics, and repeat biology.

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Father-to-offspring transmission of extremely long NOTCH2NLC repeat expansions with contractions: genetic and epigenetic profiling with long-read sequencing

Clinical Epigenetics ◽

10.1186/s13148-021-01192-5 ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

Hiromi Fukuda ◽

Daisuke Yamaguchi ◽

Kristofor Nyquist ◽

Yasushi Yabuki ◽

Satoko Miyatake ◽

...

Keyword(s):

Dna Methylation ◽

Single Molecule ◽

Dna Sequences ◽

Sequence Data ◽

Cpg Island ◽

Methylation Status ◽

Somatic Mosaicism ◽

Long Read ◽

Repeat Expansions ◽

Sporadic Cases

Abstract Background GGC repeat expansions in NOTCH2NLC are associated with neuronal intranuclear inclusion disease. Very recently, asymptomatic carriers with NOTCH2NLC repeat expansions were reported. In these asymptomatic individuals, the CpG island in NOTCH2NLC is hypermethylated, suggesting that two factors repeat length and DNA methylation status should be considered to evaluate pathogenicity. Long-read sequencing can be used to simultaneously profile genomic and epigenomic alterations. We analyzed four sporadic cases with NOTCH2NLC repeat expansion and their phenotypically normal parents. The native genomic DNA that retains base modification was sequenced on a per-trio basis using both PacBio and Oxford Nanopore long-read sequencing technologies. A custom workflow was developed to evaluate DNA modifications. With these two technologies combined, long-range DNA methylation information was integrated with complete repeat DNA sequences to investigate the genetic origins of expanded GGC repeats in these sporadic cases. Results In all four families, asymptomatic fathers had longer expansions (median: 522, 390, 528 and 650 repeats) compared with their affected offspring (median: 93, 117, 162 and 140 repeats, respectively). These expansions are much longer than the disease-causing range previously reported (in general, 41–300 repeats). Repeat lengths were extremely variable in the father, suggesting somatic mosaicism. Instability is more frequent in alleles with uninterrupted pure GGCs. Single molecule epigenetic analysis revealed complex DNA methylation patterns and epigenetic heterogeneity. We identified an aberrant gain-of-methylation region (2.2 kb in size beyond the CpG island and GGC repeats) in asymptomatic fathers. This methylated region was unmethylated in the normal allele with bilateral transitional zones with both methylated and unmethylated CpG dinucleotides, which may be protected from methylation to ensure NOTCH2NLC expression. Conclusions We clearly demonstrate that the four sporadic NOTCH2NLC-related cases are derived from the paternal GGC repeat contraction associated with demethylation. The entire genetic and epigenetic landscape of the NOTCH2NLC region was uncovered using the custom workflow of long-read sequence data, demonstrating the utility of this method for revealing epigenetic/mutational changes in repetitive elements, which are difficult to characterize by conventional short-read/bisulfite sequencing methods. Our approach should be useful for biomedical research, aiding the discovery of DNA methylation abnormalities through the entire genome.

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BleTIES: Annotation of natural genome editing in ciliates using long read sequencing

Bioinformatics ◽

10.1093/bioinformatics/btab613 ◽

2021 ◽

Author(s):

Brandon K B Seah ◽

Estienne C Swart

Keyword(s):

Dna Sequences ◽

Sequence Data ◽

Low Complexity ◽

Supplementary Information ◽

Software Toolkit ◽

Assembly Strategy ◽

Sequencing Technologies ◽

Long Reads ◽

Oxford Nanopore ◽

Long Read

Abstract Summary Ciliates are single-celled eukaryotes that eliminate specific, interspersed DNA sequences (internally eliminated sequences, IESs) from their genomes during development. These are challenging to annotate and assemble because IES-containing sequences are typically much less abundant in the cell than those without, and IES sequences themselves often contain repetitive and low-complexity sequences. Long read sequencing technologies from Pacific Biosciences and Oxford Nanopore have the potential to reconstruct longer IESs than has been possible with short reads, but require a different assembly strategy. Here we present BleTIES, a software toolkit for detecting, assembling, and analyzing IESs using mapped long reads. Availability and implementation BleTIES is implemented in Python 3. Source code is available at https://github.com/Swart-lab/bleties (MIT license), and also distributed via Bioconda. Supplementary information Benchmarking of BleTIES with published sequence data.

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Long-read, whole genome shotgun sequence data for five model organisms

10.1101/008037 ◽

2014 ◽

Cited By ~ 2

Author(s):

Kristi E Kim ◽

Paul Peluso ◽

Primo Baybayan ◽

Patricia Jane Yeadon ◽

Charles Yu ◽

...

Keyword(s):

Single Molecule ◽

De Novo ◽

Sequence Data ◽

Genome Structure ◽

Model Systems ◽

Model Organisms ◽

Biological Research ◽

Whole Genome ◽

De Novo Genome Assembly ◽

Long Read

Single molecule, real-time (SMRT) sequencing from Pacific Biosciences is increasingly used in many areas of biological research including de novo genome assembly, structural-variant identification, haplotype phasing, mRNA isoform discovery, and base-modification analyses. High-quality, public datasets of SMRT sequences can spur development of analytic tools that can accommodate unique characterisitcs of SMRT data (long read lengths, lack of GC or amplification bias, and a random error profile leading to high consensus accuracy). In this paper, we describe eight high-coverage SMRT sequence datasets from five organisms (Escherichia coli, Saccharomyces cerevisiae, Neurospora crassa, Arabidopsis thaliana, and Drosophila melanogaster) that have been publicly released to the general scientific community (NCBI Sequence Read Archive ID SRP040522). Data were generated using two sequencing chemistries (P4-C2 and P5-C3) on the PacBio RS II instrument. The datasets reported here can be used without restriction by the research community to generate whole-genome assemblies, test new algorithms, investigate genome structure and evolution, and identify base modifications in some of the most widely-studied model systems in biological research.

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