De novo identification and sequence assembly of high-copy tandem repeats in raw data Oxford Nanopore plant DNA sequencing data

The gene numbers and evolutionary rates of birds were assumed to be much lower than that of mammals, which in sharp contrast to the huge species number and morphological diversity of birds. It is very necessary to construct a complete avian genome and analyze its evolution.We constructed a chicken pan-genome from 20 de novo genome assemblies with high sequencing depth, newly identified 1,335 protein-coding genes and 3,011 long noncoding RNAs. The majority of these novel genes were detected across most individuals of the examined transcriptomes but were accidentally measured in each of the DNA sequencing data regardless of Illumina or PacBio technology. Furthermore, different from previous pan-genome models, most of these novel genes were overrepresented on chromosomal sub-telomeric regions, surrounded with extremely high proportions of tandem repeats, and strongly blocked DNA sequencing. These hidden genes were proved to be shared by all chicken genomes, included many housekeeping genes, and enriched in immune pathways. Comparative genomics revealed the novel genes had three-fold elevated substitution rates than known ones, updating the evolutionary rates of birds. Our study provides a framework for constructing a better chicken genome, which will contribute towards the understanding of avian evolution and improvement of poultry breeding.

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ntHits: de novo repeat identification of genomics data using a streaming approach

10.1101/2020.11.02.365809 ◽

2020 ◽

Author(s):

Hamid Mohamadi ◽

Justin Chu ◽

Lauren Coombe ◽

Rene Warren ◽

Inanc Birol

Keyword(s):

Dna Sequencing ◽

Large Scale ◽

De Novo ◽

Software Tool ◽

Segmental Duplications ◽

Sequencing Data ◽

Exact Methods ◽

Data Set ◽

Repeat Elements ◽

Streaming Algorithm

AbstractMotivationRepeat elements such as satellites, transposons, high number of gene copies, and segmental duplications are abundant in eukaryotic genomes. They often induce many local alignments, complicating sequence assembly and comparisons between genomes and analysis of large-scale duplications and rearrangements. Hence, identification and classification of repeats is a fundamental step in many genomics applications and their downstream analysis tools.ResultsIn this work, we present an efficient streaming algorithm and software tool, ntHits, for de novo repeat identification based on the statistical analysis of the k-mer content profile of large-scale DNA sequencing data. In the proposed algorithm, we first obtain the k-mer coverage histograms of input datasets using the ntCard algorithm, an efficient streaming algorithm for estimating the k-mer coverage histograms. From the obtained k-mer coverage histogram, the repetitive k-mers would present a long tail to the distribution of k-mer coverage profile. Experimental results show that ntHits can efficiently and accurately identify the repeat content in large-scale DNA sequencing data. For example, ntHits accurately identifies the repeat k-mers in the white spruce sequencing data set with 96× sequencing coverage in about 12 hours and using less than 150GB of memory, while using the exact methods for reporting the repeated k-mers takes several days and terabytes of memory and disk space.AvailabilityntHits is written in C++ and is released under the MIT License. It is freely available at https://github.com/bcgsc/[email protected]

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Illumina and PacBio DNA sequencing data, de novo assembly and annotation of the genome of Aurantiochytrium limacinum strain CCAP_4062/1

Data in Brief ◽

10.1016/j.dib.2020.105729 ◽

2020 ◽

Vol 31 ◽

pp. 105729 ◽

Cited By ~ 1

Author(s):

Christian Morabito ◽

Riccardo Aiese Cigliano ◽

Eric Maréchal ◽

Fabrice Rébeillé ◽

Alberto Amato

Keyword(s):

Dna Sequencing ◽

De Novo Assembly ◽

De Novo ◽

Sequencing Data

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Draft genome assemblies using sequencing reads from Oxford Nanopore Technology and Illumina platforms for four species of North American Fundulus killifish

GigaScience ◽

10.1093/gigascience/giaa067 ◽

2020 ◽

Vol 9 (6) ◽

Cited By ~ 3

Author(s):

Lisa K Johnson ◽

Ruta Sahasrabudhe ◽

James Anthony Gill ◽

Jennifer L Roach ◽

Lutz Froenicke ◽

...

Keyword(s):

North American ◽

De Novo ◽

Draft Genome ◽

Whole Genome Sequencing Data ◽

Sequencing Data ◽

Sequence Coverage ◽

Short Read ◽

Oxford Nanopore ◽

Long Read ◽

Genome Assemblies

Abstract Background Whole-genome sequencing data from wild-caught individuals of closely related North American killifish species (Fundulus xenicus, Fundulus catenatus, Fundulus nottii, and Fundulus olivaceus) were obtained using long-read Oxford Nanopore Technology (ONT) PromethION and short-read Illumina platforms. Findings Draft de novo reference genome assemblies were generated using a combination of long and short sequencing reads. For each species, the PromethION platform was used to generate 30–45× sequence coverage, and the Illumina platform was used to generate 50–160× sequence coverage. Illumina-only assemblies were fragmented with high numbers of contigs, while ONT-only assemblies were error prone with low BUSCO scores. The highest N50 values, ranging from 0.4 to 2.7 Mb, were from assemblies generated using a combination of short- and long-read data. BUSCO scores were consistently >90% complete using the Eukaryota database. Conclusions High-quality genomes can be obtained from a combination of using short-read Illumina data to polish assemblies generated with long-read ONT data. Draft assemblies and raw sequencing data are available for public use. We encourage use and reuse of these data for assembly benchmarking and other analyses.

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Assembly of the Mitochondrial Genome in the Campanulaceae Family Using Illumina Low-Coverage Sequencing

Genes ◽

10.3390/genes9080383 ◽

2018 ◽

Vol 9 (8) ◽

pp. 383 ◽

Cited By ~ 2

Author(s):

Hyun-Oh Lee ◽

Ji-Weon Choi ◽

Jeong-Ho Baek ◽

Jae-Hyeon Oh ◽

Sang-Choon Lee ◽

...

Keyword(s):

Mitochondrial Genome ◽

Dna Sequencing ◽

De Novo ◽

Genome Structure ◽

Sequencing Data ◽

Phylogenetic Characterization ◽

Low Coverage ◽

Paired End Sequencing ◽

Circular Chromosomes

Platycodon grandiflorus (balloon flower) and Codonopsis lanceolata (bonnet bellflower) are important herbs used in Asian traditional medicine, and both belong to the botanical family Campanulaceae. In this study, we designed and implemented a de novo DNA sequencing and assembly strategy to map the complete mitochondrial genomes of the first two members of the Campanulaceae using low-coverage Illumina DNA sequencing data. We produced a total of 28.9 Gb of paired-end sequencing data from the genomic DNA of P. grandiflorus (20.9 Gb) and C. lanceolata (8.0 Gb). The assembled mitochondrial genome of P. grandiflorus was found to consist of two circular chromosomes; the master circle contains 56 genes, and the minor circle contains 42 genes. The C. lanceolata mitochondrial genome consists of a single circle harboring 54 genes. Using a comparative genome structure and a pattern of repeated sequences, we show that the P. grandiflorus minor circle resulted from a recombination event involving the direct repeats of the master circle. Our dataset will be useful for comparative genomics and for evolutionary studies, and will facilitate further biological and phylogenetic characterization of species in the Campanulaceae.

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Clustering de Novo by Gene of Long Reads from Transcriptomics Data

10.1101/170035 ◽

2017 ◽

Cited By ~ 3

Author(s):

Camille Marchet ◽

Lolita Lecompte ◽

Corinne Da Silva ◽

Corinne Cruaud ◽

Jean-Marc Aury ◽

...

Keyword(s):

De Novo ◽

Free Access ◽

Sequencing Data ◽

Base Pairs ◽

Long Reads ◽

Oxford Nanopore ◽

Processing Step ◽

Whole Transcriptome Sequencing ◽

Long Read ◽

Transcriptomics Data

AbstractLong-read sequencing currently provides sequences of several thousand base pairs. This allows to obtain complete transcripts, which offers an un-precedented vision of the cellular transcriptome.However the literature is lacking tools to cluster such data de novo, in particular for Oxford Nanopore Technologies reads, because of the inherent high error rate compared to short reads.Our goal is to process reads from whole transcriptome sequencing data accurately and without a reference genome in order to reliably group reads coming from the same gene. This de novo approach is therefore particularly suitable for non-model species, but can also serve as a useful pre-processing step to improve read mapping. Our contribution is both to propose a new algorithm adapted to clustering of reads by gene and a practical and free access tool that permits to scale the complete processing of eukaryotic transcriptomes.We sequenced a mouse RNA sample using the MinION device, this dataset is used to compare our solution to other algorithms used in the context of biological clustering. We demonstrate its is better-suited for transcriptomics long reads. When a reference is available thus mapping possible, we show that it stands as an alternative method that predicts complementary clusters.

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Genome-wide profiling of heritable and de novo STR variations

10.1101/077727 ◽

2016 ◽

Cited By ~ 7

Author(s):

Thomas Willems ◽

Dina Zielinski ◽

Assaf Gordon ◽

Melissa Gymrek ◽

Yaniv Erlich

Keyword(s):

Tandem Repeats ◽

High Throughput Sequencing ◽

De Novo ◽

Genetic Diseases ◽

Whole Genome Sequencing Data ◽

Sequencing Data ◽

High Throughput Sequencing Data ◽

Genome Wide ◽

A Genome ◽

Short Tandem

AbstractShort tandem repeats (STRs) are highly variable elements that play a pivotal role in multiple genetic diseases, population genetics applications, and forensic casework. However, STRs have proven problematic to genotype from high-throughput sequencing data. Here, we describe HipSTR, a novel haplotype-based method for robustly genotyping, haplotyping, and phasing STRs from whole genome sequencing data and report a genome-wide analysis and validation of de novo STR mutations.

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tailfindr: Alignment-free poly(A) length measurement for Oxford Nanopore RNA and DNA sequencing

10.1101/588343 ◽

2019 ◽

Cited By ~ 1

Author(s):

Maximilian Krause ◽

Adnan M. Niazi ◽

Kornel Labun ◽

Yamila N. Torres Cleuren ◽

Florian S. Müller ◽

...

Keyword(s):

Dna Sequencing ◽

Nuclear Export ◽

Messenger Rna ◽

Tail Length ◽

R Package ◽

Full Length ◽

Sequencing Data ◽

Oxford Nanopore ◽

Wide Range ◽

Rna And Dna

Polyadenylation at the 3’-end is a major regulator of messenger RNA and its length is known to affect nuclear export, stability and translation, among others. Only recently, strategies have emerged that allow for genome-wide poly(A) length assessment. These methods identify genes connected to poly(A) tail measurements indirectly by short-read alignment to genetic 3’-ends. Concurrently Oxford Nanopore Technologies (ONT) established full-length isoform RNA sequencing containing the entire poly(A) tail. However, assessing poly(A) length through basecalling has so far not been possible due the inability to resolve long homopolymeric stretches in ONT sequencing.Here we presenttailfindr, an R package to estimate poly(A) tail length on ONT long-read sequencing data.tailfindroperates on unaligned, basecalled data. It measures poly(A) tail length from both native RNA and DNA sequencing, which makes poly(A) tail studies by full-length cDNA approaches possible for the first time. We assesstailfindr’sperformance across different poly(A) lengths, demonstrating thattailfindris a versatile tool providing poly(A) tail estimates across a wide range of sequencing conditions.

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Processing human frontal cortex brain tissue for population-scale Oxford Nanopore long-read DNA sequencing SOP v1

10.17504/protocols.io.b2ucqesw ◽

2021 ◽

Author(s):

Kimberley J J Billingsley ◽

Ramita Dewan ◽

Laksh Malik ◽

Pilar Alvarez Jerez ◽

Stith Kiley ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Dna Sequencing ◽

Frontal Cortex ◽

Brain Tissue ◽

Sequencing Data ◽

Oxford Nanopore ◽

Human Frontal Cortex ◽

Long Read ◽

Population Scale

Processing human frontal cortex brain tissue for population-scale Oxford Nanopore long-read DNA sequencing SOP At the NIH's Center for Alzheimer's and Related Dementias (CARD) https://card.nih.gov/research-programs/long-read-sequencing we will generate long-read sequencing data from roughly 4000 patients with Alzheimer's disease, frontotemporal dementia, Lewy body dementia, and healthy subjects. With this research, we will build a public resource consisting of long-read genome sequencing data from a large number of confirmed people with Alzheimer's disease and related dementias and healthy individuals. To generate this large-scale nanopore sequencing data we have developed a protocol for processing and long-read sequencing human frontal cortex brain tissue, targeting an N50 of ~30kb and ~30X coverage. †Correspondence to: Kimberley Billingsley [email protected] and Cornelis Blauwendraat [email protected] Acknowledgements: We would like to thank the Nanopore team (Androo Markham &Hannah Lucio), Circulomics Inc team (Jeffrey Burke, Michelle Kim, Duncan Kilburn & Kelvin Liu) and the whole CARD long-read team listed below => UCSC: Benedict Paten, Mikhail Kolmogorov, Miten Jain, Kishwar Shafin, Trevor Pesout; NHGRI: Adam Phillippy, Arang Rhie; Baylor: Fritz Sedlazeck; JHU: Winston Timp; NINDS: Sonja Scholz; NIA: Cornelis Blauwendraat, Kimberley Billingsley, Frank Grenn, Pilar Alvarez Jerez, Bryan Traynor, Shannon Ballard, Caroline Pantazis; CZI: Paolo Carnevali.

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Comparison of long-read methods for sequencing and assembly of a plant genome

GigaScience ◽

10.1093/gigascience/giaa146 ◽

2020 ◽

Vol 9 (12) ◽

Author(s):

Valentine Murigneux ◽

Subash Kumar Rai ◽

Agnelo Furtado ◽

Timothy J C Bruxner ◽

Wei Tian ◽

...

Keyword(s):

De Novo ◽

Cost Effective ◽

Genome Project ◽

Plant Genome ◽

Sequencing Data ◽

Pacific Biosciences ◽

Sequencing Technologies ◽

Oxford Nanopore ◽

Long Read ◽

The Cost

Abstract Background Sequencing technologies have advanced to the point where it is possible to generate high-accuracy, haplotype-resolved, chromosome-scale assemblies. Several long-read sequencing technologies are available, and a growing number of algorithms have been developed to assemble the reads generated by those technologies. When starting a new genome project, it is therefore challenging to select the most cost-effective sequencing technology, as well as the most appropriate software for assembly and polishing. It is thus important to benchmark different approaches applied to the same sample. Results Here, we report a comparison of 3 long-read sequencing technologies applied to the de novo assembly of a plant genome, Macadamia jansenii. We have generated sequencing data using Pacific Biosciences (Sequel I), Oxford Nanopore Technologies (PromethION), and BGI (single-tube Long Fragment Read) technologies for the same sample. Several assemblers were benchmarked in the assembly of Pacific Biosciences and Nanopore reads. Results obtained from combining long-read technologies or short-read and long-read technologies are also presented. The assemblies were compared for contiguity, base accuracy, and completeness, as well as sequencing costs and DNA material requirements. Conclusions The 3 long-read technologies produced highly contiguous and complete genome assemblies of M. jansenii. At the time of sequencing, the cost associated with each method was significantly different, but continuous improvements in technologies have resulted in greater accuracy, increased throughput, and reduced costs. We propose updating this comparison regularly with reports on significant iterations of the sequencing technologies.

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