scholarly journals Single-molecule sequencing of long DNA molecules allows high contiguity de novo genome assembly for the fungus fly, Sciara coprophila

2020 ◽  
Author(s):  
John M. Urban ◽  
Michael S. Foulk ◽  
Jacob E. Bliss ◽  
C. Michelle Coleman ◽  
Nanyan Lu ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Single Molecule ◽  
De Novo ◽  
Draft Genome ◽  
Chromosome Elimination ◽  
Future Research ◽  
Paternal Chromosome ◽  
Oxford Nanopore ◽  
Dna Modifications ◽  
Long Read

ABSTRACTThe lower Dipteran fungus fly, Sciara coprophila, has many unique biological features. For example, Sciara undergoes paternal chromosome elimination and maternal X chromosome nondisjunction during spermatogenesis, paternal X elimination during embryogenesis, intrachromosomal DNA amplification of DNA puff loci during larval development, and germline-limited chromosome elimination from all somatic cells. Paternal chromosome elimination in Sciara was the first observation of imprinting, though the mechanism remains a mystery. Here, we present the first draft genome sequence for Sciara coprophila to take a large step forward in aiding these studies. We approached assembling the Sciara genome using multiple sequencing technologies: PacBio, Oxford Nanopore MinION, and Illumina. To find an optimal assembly using these datasets, we generated 44 Illumina assemblies using 7 short-read assemblers and 50 long-read assemblies of PacBio and MinION sequence data using 6 long-read assemblers. We ranked assemblies using a battery of reference-free metrics, and scaffolded a subset of the highest-ranking assemblies using BioNano Genomics optical maps. RNA-seq datasets from multiple life stages and both sexes facilitated genome annotation. Moreover, we anchored nearly half of the Sciara genome sequence into chromosomes. Finally, we used the signal level of both the PacBio and Oxford Nanopore data to explore the presence or absence of DNA modifications in the Sciara genome since DNA modifications may play a role in imprinting in Sciara, as they do in mammals. These data serve as the foundation for future research by the growing community studying the unique features of this emerging model system.

scholarly journals High contiguity de novo genome assembly and DNA modification analyses for the fungus fly, Sciara coprophila, using single-molecule sequencing

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
John M. Urban ◽  
Michael S. Foulk ◽  
Jacob E. Bliss ◽  
C. Michelle Coleman ◽  
Nanyan Lu ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Single Molecule ◽  
De Novo ◽  
Bacterial Genome ◽  
Draft Genome ◽  
Dna Amplification ◽  
Chromosome Elimination ◽  
Paternal Chromosome ◽  
De Novo Genome Assembly ◽  
Long Read

Abstract Background The lower Dipteran fungus fly, Sciara coprophila, has many unique biological features that challenge the rule of genome DNA constancy. For example, Sciara undergoes paternal chromosome elimination and maternal X chromosome nondisjunction during spermatogenesis, paternal X elimination during embryogenesis, intrachromosomal DNA amplification of DNA puff loci during larval development, and germline-limited chromosome elimination from all somatic cells. Paternal chromosome elimination in Sciara was the first observation of imprinting, though the mechanism remains a mystery. Here, we present the first draft genome sequence for Sciara coprophila to take a large step forward in addressing these features. Results We assembled the Sciara genome using PacBio, Nanopore, and Illumina sequencing. To find an optimal assembly using these datasets, we generated 44 short-read and 50 long-read assemblies. We ranked assemblies using 27 metrics assessing contiguity, gene content, and dataset concordance. The highest-ranking assemblies were scaffolded using BioNano optical maps. RNA-seq datasets from multiple life stages and both sexes facilitated genome annotation. A set of 66 metrics was used to select the first draft assembly for Sciara. Nearly half of the Sciara genome sequence was anchored into chromosomes, and all scaffolds were classified as X-linked or autosomal by coverage. Conclusions We determined that X-linked genes in Sciara males undergo dosage compensation. An entire bacterial genome from the Rickettsia genus, a group known to be endosymbionts in insects, was co-assembled with the Sciara genome, opening the possibility that Rickettsia may function in sex determination in Sciara. Finally, the signal level of the PacBio and Nanopore data support the presence of cytosine and adenine modifications in the Sciara genome, consistent with a possible role in imprinting.

scholarly journals A draft genome sequence for the Ixodes scapularis cell line, ISE6

F1000Research ◽  
2018 ◽  
Vol 7 ◽  
pp. 297 ◽  
Author(s):  
Jason R. Miller ◽  
Sergey Koren ◽  
Kari A. Dilley ◽  
Derek M. Harkins ◽  
Timothy B. Stockwell ◽  
...  
Keyword(s):  
Cell Line ◽  
Genome Sequence ◽  
Single Molecule ◽  
Reference Genome ◽  
Sequence Data ◽  
Ixodes Scapularis ◽  
Draft Genome ◽  
Clinical Samples ◽  
Draft Assembly ◽  
Long Read

Background:The tick cell line ISE6, derived fromIxodes scapularis, is commonly used for amplification and detection of arboviruses in environmental or clinical samples.Methods:To assist with sequence-based assays, we sequenced the ISE6 genome with single-molecule, long-read technology.Results:The draft assembly appears near complete based on gene content analysis, though it appears to lack some instances of repeats in this highly repetitive genome. The assembly appears to have separated the haplotypes at many loci. DNA short read pairs, used for validation only, mapped to the cell line assembly at a higher rate than they mapped to theIxodes scapularisreference genome sequence.Conclusions:The assembly could be useful for filtering host genome sequence from sequence data obtained from cells infected with pathogens.

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

GigaScience ◽  
2020 ◽  
Vol 9 (6) ◽  
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.

scholarly journals Genomic Surveillance for Antimicrobial Resistance inMannheimia haemolyticaUsing Nanopore Single Molecule Sequencing Technology

2018 ◽  
Author(s):  
Alexander Lim ◽  
Bryan Naidenov ◽  
Haley Bates ◽  
Karyn Willyerd ◽  
Timothy Snider ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Antimicrobial Resistance ◽  
Single Molecule ◽  
Resistant Strain ◽  
De Novo ◽  
Gene Annotation ◽  
Cost Effective ◽  
Multidrug Resistant ◽  
Oxford Nanopore ◽  
Long Read

AbstractDisruptive innovations in long-range, cost-effective direct template nucleic acid sequencing are transforming clinical and diagnostic medicine. A multidrug resistant strain and a pan-susceptible strain ofMannheimia haemolytica, isolated from pneumonic bovine lung samples, were respectively sequenced at 146x and 111x coverage with Oxford Nanopore Technologies MinION.De novoassembly produced a complete genome for the non-resistant strain and a nearly complete assembly for the drug resistant strain. Functional annotation using RAST (Rapid Annotations using Subsystems Technology), CARD (Comprehensive Antibiotic Resistance Database) and ResFinder databases identified genes conferring resistance to different classes of antibiotics including beta lactams, tetracyclines, lincosamides, phenicols, aminoglycosides, sulfonamides and macrolides. Antibiotic resistance phenotypes of theM. haemolyticastrains were confirmed with minimum inhibitory concentration (MIC) assays. The sequencing capacity of highly portable MinION devices was verified by sub-sampling sequencing reads; potential for antimicrobial resistance determined by identification of resistance genes in the draft assemblies with as little as 5,437 MinION reads corresponded to all classes of MIC assays. The resulting quality assemblies and AMR gene annotation highlight efficiency of ultra long-read, whole-genome sequencing (WGS) as a valuable tool in diagnostic veterinary medicine.

scholarly journals Genome sequence analysis of a giant-rooted ‘Sakurajima daikon’ radish (Raphanus sativus)

2020 ◽  
Author(s):  
Kenta Shirasawa ◽  
Hideki Hirakawa ◽  
Nobuko Fukino ◽  
Hiroyasu Kitashiba ◽  
Sachiko Isobe
Keyword(s):  
Genome Sequence ◽  
Single Molecule ◽  
Raphanus Sativus ◽  
Molecular Mechanisms ◽  
De Novo ◽  
Genetic Maps ◽  
Complex Nature ◽  
Eukaryotic Genes ◽  
Long Read ◽  
Large Round

AbstractDaikon radish (Raphanus sativus) roots vary in size and shape between cultivars. This study reports the genome sequence assembly of a giant-rooted ‘Sakurajima daikon’ radish variety, ‘Okute-Sakurajima’, which produces extremely large round roots. Radish genome assembly is hampered by the repetitive and complex nature of the genome. To address this, single-molecule real-time technology was used to obtain long-read sequences at 60× genome coverage. De novo assembly of the long reads generated 504.5 Mb contig sequences consisting of 1,437 sequences with contig N50 length of 1.2 Mb, including 94.1% of the core eukaryotic genes. Nine pseudomolecule sequences, comprising 69.3% of the assembled contig length, were generated with high-density SNP genetic maps. The chromosome-level sequences revealed structure variations and rearrangements among Brassicaceae genomes. In total, 89,915 genes were predicted in the ‘Okute-Sakurajima’ genome, 30,033 of which were unique to the assembly in this study. The improved genome information generated in this study will not only form a new baseline resource for radish genomics, but will also provide insights into the molecular mechanisms underlying formation of giant radish roots.

scholarly journals lra: A long read aligner for sequences and contigs

2021 ◽  
Vol 17 (6) ◽  
pp. e1009078
Author(s):  
Jingwen Ren ◽  
Mark J. P. Chaisson
Keyword(s):  
Dynamic Programming ◽  
Single Molecule ◽  
De Novo Assembly ◽  
De Novo ◽  
Concave Function ◽  
Single Molecule Sequencing ◽  
Link Type ◽  
Oxford Nanopore ◽  
Concave Cost ◽  
Long Read

It is computationally challenging to detect variation by aligning single-molecule sequencing (SMS) reads, or contigs from SMS assemblies. One approach to efficiently align SMS reads is sparse dynamic programming (SDP), where optimal chains of exact matches are found between the sequence and the genome. While straightforward implementations of SDP penalize gaps with a cost that is a linear function of gap length, biological variation is more accurately represented when gap cost is a concave function of gap length. We have developed a method, lra, that uses SDP with a concave-cost gap penalty, and used lra to align long-read sequences from PacBio and Oxford Nanopore (ONT) instruments as well as de novo assembly contigs. This alignment approach increases sensitivity and specificity for SV discovery, particularly for variants above 1kb and when discovering variation from ONT reads, while having runtime that are comparable (1.05-3.76×) to current methods. When applied to calling variation from de novo assembly contigs, there is a 3.2% increase in Truvari F1 score compared to minimap2+htsbox. lra is available in bioconda (https://anaconda.org/bioconda/lra) and github (https://github.com/ChaissonLab/LRA).

scholarly journals Draft genome sequence of the pulse crop blackgram [Vigna mungo (L.) Hepper] reveals potential R-genes

2020 ◽  
Author(s):  
J Souframanien ◽  
Avi Raizada ◽  
Punniyamoorthy Dhanasekar ◽  
Penna Suprasanna
Keyword(s):  
Genome Sequence ◽  
De Novo ◽  
Draft Genome ◽  
Vigna Mungo ◽  
Draft Genome Sequence ◽  
Sequence Length ◽  
Legume Crop ◽  
Oxford Nanopore ◽  
Hybrid Genome ◽  
First Time

AbstractBlackgram [Vigna mungo (L.) Hepper] (2n = 2x = 22), an important Asiatic legume crop, is a major source of dietary protein for the predominantly vegetarian population. Here we construct a draft genome sequence of blackgram, for the first time, by employing hybrid genome assembly with Illumina reads and third generation Oxford Nanopore sequencing technology. The final de novo whole genome of blackgram is ~ 475 Mb (82 % of the genome) and has maximum scaffold length of 6.3 Mb with scaffold N50 of1.42 Mb. Genome analysis identified 18655 genes with mean coding sequence length of 970bp. Around 96.7 % of predicted genes were annotated. Nearly half of the assembled sequence is composed of repetitive elements with retrotransposons as major (47.3% of genome) transposable elements, whereas, DNA transposons made up only 2.29% of the genome. A total of 166014 SSRs, including 65180 compound SSRs, were identified and primer pairs for 34816 SSRs were designed. Out of the 18665 proteins, 678 proteins showed presence of R-gene related domains. KIN class was found in majority of the proteins (372) followed by RLK (79) and N (79). The genome sequence of blackgram will facilitate identification of agronomically important genes and accelerate the genetic improvement of blackgram.

scholarly journals Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation

2016 ◽  
Author(s):  
Sergey Koren ◽  
Brian P. Walenz ◽  
Konstantin Berlin ◽  
Jason R. Miller ◽  
Nicholas H. Bergman ◽  
...  
Keyword(s):  
Single Molecule ◽  
De Novo ◽  
Error Rates ◽  
Celera Assembler ◽  
Oxford Nanopore ◽  
Long Read ◽  
Reference Quality ◽  
Order Of Magnitude ◽  
Assembly Algorithms ◽  
Oxford Nanopore Technologies

AbstractLong-read single-molecule sequencing has revolutionized de novo genome assembly and enabled the automated reconstruction of reference-quality genomes. However, given the relatively high error rates of such technologies, efficient and accurate assembly of large repeats and closely related haplotypes remains challenging. We address these issues with Canu, a successor of Celera Assembler that is specifically designed for noisy single-molecule sequences. Canu introduces support for nanopore sequencing, halves depth-of-coverage requirements, and improves assembly continuity while simultaneously reducing runtime by an order of magnitude on large genomes versus Celera Assembler 8.2. These advances result from new overlapping and assembly algorithms, including an adaptive overlapping strategy based on tf-idf weighted MinHash and a sparse assembly graph construction that avoids collapsing diverged repeats and haplotypes. We demonstrate that Canu can reliably assemble complete microbial genomes and near-complete eukaryotic chromosomes using either PacBio or Oxford Nanopore technologies, and achieves a contig NG50 of greater than 21 Mbp on both human and Drosophila melanogaster PacBio datasets. For assembly structures that cannot be linearly represented, Canu provides graph-based assembly outputs in graphical fragment assembly (GFA) format for analysis or integration with complementary phasing and scaffolding techniques. The combination of such highly resolved assembly graphs with long-range scaffolding information promises the complete and automated assembly of complex genomes.

scholarly journals Draft genome sequence of the pulse crop blackgram [Vigna mungo (L.) Hepper] reveals potential R-genes

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Souframanien Jegadeesan ◽  
Avi Raizada ◽  
Punniyamoorthy Dhanasekar ◽  
Penna Suprasanna
Keyword(s):  
Genome Sequence ◽  
De Novo ◽  
Draft Genome ◽  
Vigna Mungo ◽  
Draft Genome Sequence ◽  
Sequence Length ◽  
Legume Crop ◽  
Oxford Nanopore ◽  
Hybrid Genome ◽  
First Time

AbstractBlackgram [Vigna mungo (L.) Hepper] (2n = 2x = 22), an important Asiatic legume crop, is a major source of dietary protein for the predominantly vegetarian population. Here we construct a draft genome sequence of blackgram, for the first time, by employing hybrid genome assembly with Illumina reads and third generation Oxford Nanopore sequencing technology. The final de novo whole genome of blackgram is ~ 475 Mb (82% of the genome) and has maximum scaffold length of 6.3 Mb with scaffold N50 of 1.42 Mb. Genome analysis identified 42,115 genes with mean coding sequence length of 1131 bp. Around 80.6% of predicted genes were annotated. Nearly half of the assembled sequence is composed of repetitive elements with retrotransposons as major (47.3% of genome) transposable elements, whereas, DNA transposons made up only 2.29% of the genome. A total of 166,014 SSRs, including 65,180 compound SSRs, were identified and primer pairs for 34,816 SSRs were designed. Out of the 33,959 proteins, 1659 proteins showed presence of R-gene related domains. KIN class was found in majority of the proteins (905) followed by RLK (239) and RLP (188). The genome sequence of blackgram will facilitate identification of agronomically important genes and accelerate the genetic improvement of blackgram.

scholarly journals lra: the Long Read Aligner for Sequences and Contigs

2020 ◽  
Author(s):  
Jingwen Ren ◽  
Mark JP Chaisson
Keyword(s):  
Single Molecule ◽  
De Novo ◽  
Data Types ◽  
Single Molecule Sequencing ◽  
Detection Algorithms ◽  
Link Type ◽  
Long Reads ◽  
Oxford Nanopore ◽  
Long Read ◽  
Linear Cost

AbstractMotivationIt is computationally challenging to detect variation by aligning long reads from single-molecule sequencing (SMS) instruments, or megabase-scale contigs from SMS assemblies. One approach to efficiently align long sequences is sparse dynamic programming (SDP), where exact matches are found between the sequence and the genome, and optimal chains of matches are found representing a rough alignment. Sequence variation is more accurately modeled when alignments are scored with a gap penalty that is a convex function of the gap length. Because previous implementations of SDP used a linear-cost gap function that does not accurately model variation, and implementations of alignment that have a convex gap penalty are either inefficient or use heuristics, we developed a method, lra, that uses SDP with a convex-cost gap penalty. We use lra to align long-read sequences from PacBio and Oxford Nanopore (ONT) instruments as well as de novo assembly contigs.ResultsAcross all data types, the runtime of lra is between 52-168% of the state of the art aligner minimap2 when generating SAM alignment, and 9-15% of an alternative method, ngmlr. This alignment approach may be used to provide additional evidence of SV calls in PacBio datasets, and an increase in sensitivity and specificity on ONT data with current SV detection algorithms. The number of calls discovered using pbsv with lra alignments are within 98.3-98.6% of calls made from minimap2 alignments on the same data, and give a nominal 0.2-0.4% increase in F1 score by Truvari analysis. On ONT data with SV called using Sniffles, the number of calls made from lra alignments is 3% greater than minimap2-based calls, and 30% greater than ngmlr based calls, with a 4.6-5.5% increase in Truvari F1 score. When applied to calling variation from de novo assembly contigs, there is a 5.8% increase in SV calls compared to minimap2+paftools, with a 4.3% increase in Truvari F1 score.Availability and implementationAvailable in bioconda: https://anaconda.org/bioconda/lra and github: https://github.com/ChaissonLab/[email protected], [email protected]

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