Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data

ABSTRACTHere we report genome assemblies for three strains of Wolbachia pipientis assembled from unenriched, unfiltered long-read shotgun sequencing data of geographically distinct strains of Drosophila melanogaster. Our simple methodology can be applied to long-read datasets of other Wolbachia-infected species to produce complete assemblies for this important model symbiont.

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Identifying contamination with advanced visualization and analysis practices: metagenomic approaches for eukaryotic genome assemblies

PeerJ ◽

10.7717/peerj.1839 ◽

2016 ◽

Vol 4 ◽

pp. e1839 ◽

Cited By ~ 57

Author(s):

Tom O. Delmont ◽

A. Murat Eren

Keyword(s):

High Throughput Sequencing ◽

Draft Genome ◽

Cost Effective ◽

Single Copy ◽

Eukaryotic Genome ◽

Sequencing Data ◽

Bacterial Genomes ◽

Long Read ◽

Domains Of Life ◽

Genome Assemblies

High-throughput sequencing provides a fast and cost-effective mean to recover genomes of organisms from all domains of life. However, adequate curation of the assembly results against potential contamination of non-target organisms requires advanced bioinformatics approaches and practices. Here, we re-analyzed the sequencing data generated for the tardigradeHypsibius dujardini,and created a holistic display of the eukaryotic genome assembly using DNA data originating from two groups and eleven sequencing libraries. By using bacterial single-copy genes, k-mer frequencies, and coverage values of scaffolds we could identify and characterize multiple near-complete bacterial genomes from the raw assembly, and curate a 182 Mbp draft genome forH. dujardinisupported by RNA-Seq data. Our results indicate that most contaminant scaffolds were assembled from Moleculo long-read libraries, and most of these contaminants have differed between library preparations. Our re-analysis shows that visualization and curation of eukaryotic genome assemblies can benefit from tools designed to address the needs of today’s microbiologists, who are constantly challenged by the difficulties associated with the identification of distinct microbial genomes in complex environmental metagenomes.

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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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The Genome Sequence of the Citrus Melanose Pathogen Diaporthe citri and Two Citrus related Diaporthe species

Phytopathology ◽

10.1094/phyto-08-20-0376-sc ◽

2020 ◽

Author(s):

Yunpeng Gai ◽

Tao Xiong ◽

Xiaoe Xiao ◽

Pudong Li ◽

Yating Zeng ◽

...

Keyword(s):

Molecular Diagnostic ◽

Sequencing Data ◽

Genome Sequences ◽

Protein Coding ◽

Non Profit ◽

Host Interaction ◽

Fungal Biology ◽

Long Read ◽

Genome Assemblies ◽

Paired End Sequencing

Melanose disease is one the most widely distributed and economically important fungal diseases of citrus worldwide. The causative agent is the filamentous fungus Diaporthe citri Wolf (syn. Phomopsis citri H.S. Fawc.). Here, we report the genome assemblies of three strains of D. citri, namely strains ZJUD2, ZJUD14 and Q7, which were generated using a combination of PacBio Sequel long-read and Illumina paired-end sequencing data. The assembled genomes of D. citri ranged from 52.06 Mb to 63.61 Mb in genome size, containing 15,977 ~ 16,622 protein-coding genes. We also sequenced and annotated the genome sequences of two Citrus-related Diaporthe species, D. citriasiana and D. citrichinensis. In addition, a database for citrus-related Diaporthe genomes was established to provide a public platform to access genome sequences, genome annotation and comparative genomics data of these Diaporthe species. The described genome sequences and the citrus-related Diaporthe genomes database provide a useful resource for the study of fungal biology, pathogen-host interaction, molecular diagnostic marker development, and population genomic analyses of Diaporthe species. The database will be updated regularly when the genomes of newly isolated Diaporthe species are sequenced. The citrus-related Diaporthe genomes database is freely available for non-profit use at http://www.zjudata.com/blast/diaporthe.php.

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Comparison of long read sequencing technologies in resolving bacteria and fly genomes

10.1101/2020.07.21.213975 ◽

2020 ◽

Author(s):

Eric S. Tvedte ◽

Mark Gasser ◽

Benjamin C. Sparklin ◽

Jane Michalski ◽

Xuechu Zhao ◽

...

Keyword(s):

Genome Sequencing ◽

Bacterial Genome ◽

Fruit Fly ◽

Drosophila Ananassae ◽

Whole Genome Sequencing Data ◽

Sequencing Data ◽

E Coli ◽

Hybrid Approaches ◽

Long Read ◽

Genome Assemblies

ABSTRACTBackgroundThe newest generation of DNA sequencing technology is highlighted by the ability to sequence reads hundreds of kilobases in length, and the increased availability of long read data has democratized the genome sequencing and assembly process. PacBio and Oxford Nanopore Technologies (ONT) have pioneered competitive long read platforms, with more recent work focused on improving sequencing throughput and per-base accuracy. Released in 2019, the PacBio Sequel II platform advertises substantial enhancements over previous PacBio systems.ResultsWe used whole-genome sequencing data produced by two PacBio platforms (Sequel II and RS II) and two ONT protocols (Rapid Sequencing and Ligation Sequencing) to compare assemblies of the bacteria Escherichia coli and the fruit fly Drosophila ananassae. Sequel II assemblies had higher contiguity and consensus accuracy relative to other methods, even after accounting for differences in sequencing throughput. ONT RAPID libraries had the fewest chimeric reads in addition to superior quantification of E. coli plasmids versus ligation-based libraries. The quality of assemblies can be enhanced by adopting hybrid approaches using Illumina libraries for bacterial genome assemblies or combined ONT and Sequel II libraries for eukaryotic genome assemblies. Genome-wide DNA methylation could be detected using both technologies, however ONT libraries enabled the identification of a broader range of known E. coli methyltransferase recognition motifs in addition to undocumented D. ananassae motifs.ConclusionsThe ideal choice of long read technology may depend on several factors including the question or hypothesis under examination. No single technology outperformed others in all metrics examined.

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Identifying contamination with advanced visualization and analysis practices: metagenomic approaches for eukaryotic genome assemblies

10.7287/peerj.preprints.1695 ◽

2016 ◽

Author(s):

Tom O Delmont ◽

A. Murat Eren

Keyword(s):

High Throughput Sequencing ◽

Draft Genome ◽

Cost Effective ◽

Single Copy ◽

Eukaryotic Genome ◽

Metagenomic Data ◽

Sequencing Data ◽

Long Read ◽

Domains Of Life ◽

Genome Assemblies

High-throughput sequencing provides a fast and cost effective mean to recover genomes of organisms from all domains of life. However, adequate curation of the assembly results against potential contamination of non-target organisms requires advanced bioinformatics approaches and practices. Here, we re-analyzed the sequencing data generated for the tardigrade Hypsibius dujardini using approaches routinely employed by microbial ecologists who reconstruct bacterial and archaeal genomes from metagenomic data. We created a holistic display of the eukaryotic genome assembly using DNA data originating from two groups and eleven sequencing libraries. By using bacterial single-copy genes, k-mer frequencies, and coverage values of scaffolds we could identify and characterize multiple near-complete bacterial genomes, and curate a 182 Mbp draft genome for H. dujardini supported by RNA-Seq data. Our results indicate that most contaminant scaffolds were assembled from Moleculo long-read libraries, and most of these contaminants have differed between library preparations. Our re-analysis shows that visualization and curation of eukaryotic genome assemblies can benefit from tools designed to address the needs of today’s microbiologists, who are constantly challenged by the difficulties associated with the identification of distinct microbial genomes in complex environmental metagenomes.

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WENGAN: Efficient and high quality hybrid de novo assembly of human genomes

10.1101/840447 ◽

2019 ◽

Cited By ~ 1

Author(s):

Alex Di Genova ◽

Elena Buena-Atienza ◽

Stephan Ossowski ◽

Marie-France Sagot

Keyword(s):

De Novo ◽

Computational Cost ◽

Sequence Information ◽

Sequencing Data ◽

High Quality ◽

Sequencing Technologies ◽

Human Genomes ◽

Long Reads ◽

Long Read ◽

Genome Assemblies

The continuous improvement of long-read sequencing technologies along with the development of ad-doc algorithms has launched a new de novo assembly era that promises high-quality genomes. However, it has proven difficult to use only long reads to generate accurate genome assemblies of large, repeat-rich human genomes. To date, most of the human genomes assembled from long error-prone reads add accurate short reads to further polish the consensus quality. Here, we report the development of a novel algorithm for hybrid assembly, WENGAN, and the de novo assembly of four human genomes using a combination of sequencing data generated on ONT PromethION, PacBio Sequel, Illumina and MGI technology. WENGAN implements efficient algorithms that exploit the sequence information of short and long reads to tackle assembly contiguity as well as consensus quality. The resulting genome assemblies have high contiguity (contig NG50:16.67-62.06 Mb), few assembly errors (contig NGA50:10.9-45.91 Mb), good consensus quality (QV:27.79-33.61), and high gene completeness (BUSCO complete: 94.6-95.1%), while consuming low computational resources (CPU hours:153-1027). In particular, the WENGAN assembly of the haploid CHM13 sample achieved a contig NG50 of 62.06 Mb (NGA50:45.91 Mb), which surpasses the contiguity of the current human reference genome (GRCh38 contig NG50:57.88 Mb). Providing highest quality at low computational cost, WENGAN is an important step towards the democratization of the de novo assembly of human genomes. The WENGAN assembler is available at https://github.com/adigenova/wengan

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Identifying contamination with advanced visualization and analysis practices: metagenomic approaches for eukaryotic genome assemblies

10.7287/peerj.preprints.1695v1 ◽

2016 ◽

Cited By ~ 1

Author(s):

Tom O Delmont ◽

A. Murat Eren

Keyword(s):

High Throughput Sequencing ◽

Draft Genome ◽

Cost Effective ◽

Single Copy ◽

Eukaryotic Genome ◽

Metagenomic Data ◽

Sequencing Data ◽

Long Read ◽

Domains Of Life ◽

Genome Assemblies

High-throughput sequencing provides a fast and cost effective mean to recover genomes of organisms from all domains of life. However, adequate curation of the assembly results against potential contamination of non-target organisms requires advanced bioinformatics approaches and practices. Here, we re-analyzed the sequencing data generated for the tardigrade Hypsibius dujardini using approaches routinely employed by microbial ecologists who reconstruct bacterial and archaeal genomes from metagenomic data. We created a holistic display of the eukaryotic genome assembly using DNA data originating from two groups and eleven sequencing libraries. By using bacterial single-copy genes, k-mer frequencies, and coverage values of scaffolds we could identify and characterize multiple near-complete bacterial genomes, and curate a 182 Mbp draft genome for H. dujardini supported by RNA-Seq data. Our results indicate that most contaminant scaffolds were assembled from Moleculo long-read libraries, and most of these contaminants have differed between library preparations. Our re-analysis shows that visualization and curation of eukaryotic genome assemblies can benefit from tools designed to address the needs of today’s microbiologists, who are constantly challenged by the difficulties associated with the identification of distinct microbial genomes in complex environmental metagenomes.

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Phased Haplotype Resolution of the SLC6A4 Promoter Using Long-Read Single Molecule Real-Time (SMRT) Sequencing

Genes ◽

10.3390/genes11111333 ◽

2020 ◽

Vol 11 (11) ◽

pp. 1333

Author(s):

Mariana R. Botton ◽

Yao Yang ◽

Erick R. Scott ◽

Robert J. Desnick ◽

Stuart A. Scott

Keyword(s):

Real Time ◽

Single Molecule ◽

Variable Number ◽

Sequencing Data ◽

Smrt Sequencing ◽

Short Read ◽

Short Read Sequencing ◽

Sequencing Method ◽

Long Read ◽

S Allele

The SLC6A4 gene has been implicated in psychiatric disorder susceptibility and antidepressant response variability. The SLC6A4 promoter is defined by a variable number of homologous 20–24 bp repeats (5-HTTLPR), and long (L) and short (S) alleles are associated with higher and lower expression, respectively. However, this insertion/deletion variant is most informative when considered as a haplotype with the rs25531 and rs25532 variants. Therefore, we developed a long-read single molecule real-time (SMRT) sequencing method to interrogate the SLC6A4 promoter region. A total of 120 samples were subjected to SLC6A4 long-read SMRT sequencing, primarily selected based on available short-read sequencing data. Short-read genome sequencing from the 1000 Genomes (1KG) Project (~5X) and the Genetic Testing Reference Material Coordination Program (~45X), as well as high-depth short-read capture-based sequencing (~330X), could not identify the 5-HTTLPR short (S) allele, nor could short-read sequencing phase any identified variants. In contrast, long-read SMRT sequencing unambiguously identified the 5-HTTLPR short (S) allele (frequency of 0.467) and phased SLC6A4 promoter haplotypes. Additionally, discordant rs25531 genotypes were reviewed and determined to be short-read errors. Taken together, long-read SMRT sequencing is an innovative and robust method for phased resolution of the SLC6A4 promoter, which could enable more accurate pharmacogenetic testing for both research and clinical applications.

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Contiguous and accurate de novo assembly of metazoan genomes with modest long read coverage

10.1101/029306 ◽

2015 ◽

Cited By ~ 7

Author(s):

Mahul Chakraborty ◽

James G. Baldwin-Brown ◽

Anthony D. Long ◽

J.J. Emerson

Keyword(s):

Best Practices ◽

Dna Isolation ◽

Reference Genome ◽

De Novo ◽

Error Rates ◽

Read Coverage ◽

Sequencing Data ◽

Sequencing Coverage ◽

Long Read ◽

Genome Assemblies

AbstractGenome assemblies that are accurate, complete, and contiguous are essential for identifying important structural and functional elements of genomes and for identifying genetic variation. Nevertheless, most recent genome assemblies remain incomplete and fragmented. While long molecule sequencing promises to deliver more complete genome assemblies with fewer gaps, concerns about error rates, low yields, stringent DNA requirements, and uncertainty about best practices may discourage many investigators from adopting this technology. Here, in conjunction with the platinum standard Drosophila melanogaster reference genome, we analyze recently published long molecule sequencing data to identify what governs completeness and contiguity of genome assemblies. We also present a hybrid meta-assembly approach that achieves remarkable assembly contiguity for both Drosophila and human assemblies with only modest long molecule sequencing coverage. Our results motivate a set of preliminary best practices for obtaining accurate and contiguous assemblies, a “missing manual” that guides key decisions in building high quality de novo genome assemblies, from DNA isolation to polishing the assembly.

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