scholarly journals Long-read assembly and comparative evidence-based reanalysis of Cryptosporidium genome sequences reveals expanded transporter repertoire and duplication of entire chromosome ends including subtelomeric regions

2021 ◽  
pp. gr.275325.121
Author(s):  
Rodrigo P. Baptista ◽  
Yiran Li ◽  
Adam Sateriale ◽  
Karen L. Brooks ◽  
Alan Tracey ◽  
...  
Keyword(s):  
Genome Assembly ◽  
Reference Genome ◽  
Diarrheal Disease ◽  
Gene Copy ◽  
Future Research ◽  
Single Nucleotide Variants ◽  
Cryptosporidium Hominis ◽  
Entire Chromosome ◽  
Long Read ◽  
Subtelomeric Regions

Cryptosporidiosis is a leading cause of waterborne diarrheal disease globally and an important contributor to mortality in infants and the immunosuppressed. Despite its importance, the Cryptosporidium community has only had access to a good, but incomplete, Cryptosporidium parvum IOWA reference genome sequence. Incomplete reference sequences hamper annotation, experimental design and interpretation. We have generated a new C. parvum IOWA genome assembly supported by PacBio and Oxford Nanopore long-read technologies and a new comparative and consistent genome annotation for three closely related species C. parvum, Cryptosporidium hominis and Cryptosporidium tyzzeri. We made 1,926 C. parvum annotation updates based on experimental evidence. They include new transporters, ncRNAs, introns and altered gene structures. The new assembly and annotation revealed a complete Dnmt2 methylase ortholog. Comparative annotation between C. parvum, C. hominis and C. tyzzeri revealed that most "missing" orthologs are found suggesting that the biological differences between the species must result from gene copy number variation, differences in gene regulation and single nucleotide variants (SNVs). Using the new assembly and annotation as reference, 190 genes are identified as evolving under positive selection, including many not detected previously. The new C. parvum IOWA reference genome assembly is larger, gap free and lacks ambiguous bases. This chromosomal assembly recovers all 16 chromosome ends, 13 of which are contiguously assembled. The three remaining chromosome ends are provisionally placed. These ends represent duplication of entire chromosome ends including subtelomeric regions revealing a new level of genome plasticity that will both inform and impact future research.

scholarly journals Long-read assembly and comparative evidence-based reanalysis of Cryptosporidium genome sequences reveal new biological insights

2021 ◽  
Author(s):  
Rodrigo P. Baptista ◽  
Yiran Li ◽  
Adam Sateriale ◽  
Mandy J. Sanders ◽  
Karen L. Brooks ◽  
...  
Keyword(s):  
Genome Assembly ◽  
Reference Genome ◽  
Diarrheal Disease ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Future Research ◽  
Gene Copy Number Variation ◽  
Long Read ◽  
Number Variation ◽  
Gene Structures

ABSTRACTCryptosporidiosis is a leading cause of waterborne diarrheal disease globally and an important contributor to mortality in infants and the immunosuppressed. Despite its importance, the Cryptosporidium community still relies on a fragmented reference genome sequence from 2004. Incomplete reference sequences hamper experimental design and interpretation. We have generated a new C. parvum IOWA genome assembly supported by PacBio and Oxford Nanopore long-read technologies and a new comparative and consistent genome annotation for three closely related species C. parvum, C. hominis and C. tyzzeri. The new C. parvum IOWA reference genome assembly is larger, gap free and lacks ambiguous bases. This chromosomal assembly recovers 13 of 16 possible telomeres and raises a new hypothesis for the remaining telomeres and associated subtelomeric regions. Comparative annotation revealed that most “missing” orthologs are found suggesting that species differences result primarily from structural rearrangements, gene copy number variation and SNVs in C. parvum, C. hominis and C. tyzzeri. We made >1,500 C. parvum annotation updates based on experimental evidence. They included new transporters, ncRNAs, introns and altered gene structures. The new assembly and annotation revealed a complete DNA methylase Dnmt2 ortholog. 190 genes under positive selection including many new candidates were identified using the new assembly and annotation as reference. Finally, possible subtelomeric amplification and variation events in C. parvum are detected that reveal a new level of genome plasticity that will both inform and impact future research.

scholarly journals Fully phased human genome assembly without parental data using single-cell strand sequencing and long reads

2020 ◽  
Author(s):  
David Porubsky ◽  
◽  
Peter Ebert ◽  
Peter A. Audano ◽  
Mitchell R. Vollger ◽  
...  
Keyword(s):  
Single Cell ◽  
Genome Assembly ◽  
De Novo ◽  
Error Rates ◽  
Sequencing Data ◽  
Single Nucleotide Variants ◽  
De Novo Genome Assembly ◽  
Parental Data ◽  
Human Genome Assembly ◽  
Long Read

AbstractHuman genomes are typically assembled as consensus sequences that lack information on parental haplotypes. Here we describe a reference-free workflow for diploid de novo genome assembly that combines the chromosome-wide phasing and scaffolding capabilities of single-cell strand sequencing1,2 with continuous long-read or high-fidelity3 sequencing data. Employing this strategy, we produced a completely phased de novo genome assembly for each haplotype of an individual of Puerto Rican descent (HG00733) in the absence of parental data. The assemblies are accurate (quality value > 40) and highly contiguous (contig N50 > 23 Mbp) with low switch error rates (0.17%), providing fully phased single-nucleotide variants, indels and structural variants. A comparison of Oxford Nanopore Technologies and Pacific Biosciences phased assemblies identified 154 regions that are preferential sites of contig breaks, irrespective of sequencing technology or phasing algorithms.

scholarly journals RefKA: A fast and efficient long-read genome assembly approach for large and complex genomes

2020 ◽  
Author(s):  
Yuxuan Yuan ◽  
Philipp E. Bayer ◽  
Robyn Anderson ◽  
HueyTyng Lee ◽  
Chon-Kit Kenneth Chan ◽  
...  
Keyword(s):  
Genome Assembly ◽  
Chinese Spring ◽  
Complete Genome ◽  
Reference Genome ◽  
Computing Time ◽  
Link Type ◽  
Recent Advances ◽  
Long Read ◽  
Genome Assemblies

AbstractRecent advances in long-read sequencing have the potential to produce more complete genome assemblies using sequence reads which can span repetitive regions. However, overlap based assembly methods routinely used for this data require significant computing time and resources. Here, we have developed RefKA, a reference-based approach for long read genome assembly. This approach relies on breaking up a closely related reference genome into bins, aligning k-mers unique to each bin with PacBio reads, and then assembling each bin in parallel followed by a final bin-stitching step. During benchmarking, we assembled the wheat Chinese Spring (CS) genome using publicly available PacBio reads in parallel in 168 wall hours on a 250 CPU system. The maximum RAM used was 300 Gb and the computing time was 42,000 CPU hours. The approach opens applications for the assembly of other large and complex genomes with much-reduced computing requirements. The RefKA pipeline is available at https://github.com/AppliedBioinformatics/RefKA

scholarly journals Long read sequencing reveals Poxvirus evolution through rapid homogenization of gene arrays

2018 ◽  
Author(s):  
Thomas A. Sasani ◽  
Kelsey R. Cone ◽  
Aaron R. Quinlan ◽  
Nels C. Elde
Keyword(s):  
Copy Number ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Single Nucleotide Variants ◽  
Sequencing Platform ◽  
Sequencing Technologies ◽  
Gene Copy Number Variation ◽  
Large Gene ◽  
Long Read ◽  
Number Variation

AbstractLarge DNA viruses rapidly evolve to defeat host defenses. Poxvirus adaptation can involve combinations of recombination-driven gene copy number variation and beneficial single nucleotide variants (SNVs) at the same locus, yet how these distinct mechanisms of genetic diversification might simultaneously facilitate adaptation to immune blocks is unknown. We performed experimental evolution with a vaccinia virus population harboring a SNV in a gene actively undergoing copy number amplification. Comparisons of virus genomes using the Oxford Nanopore Technologies sequencing platform allowed us to phase SNVs within large gene copy arrays for the first time, and uncovered a mechanism of adaptive SNV homogenization reminiscent of gene conversion, which is actively driven by selection. Our work reveals a new mechanism for the fluid gain of beneficial mutations in genetic regions undergoing active recombination in viruses, and illustrates the value of long read sequencing technologies for investigating complex genome dynamics in diverse biological systems.

scholarly journals Construction of a new chromosome-scale, long-read reference genome assembly of the Syrian hamster, Mesocricetus auratus

2021 ◽  
Author(s):  
R. Alan Harris ◽  
Muthuswamy Raveendran ◽  
Dustin T Lyfoung ◽  
Fritz J Sedlazeck ◽  
Medhat Mahmoud ◽  
...  
Keyword(s):  
Genome Assembly ◽  
Syrian Hamster ◽  
Reference Genome ◽  
Sequence Data ◽  
Mesocricetus Auratus ◽  
Protein Coding ◽  
Protein Coding Genes ◽  
Sequencing Technologies ◽  
Long Read ◽  
Short Read Sequence

Background The Syrian hamster (Mesocricetus auratus) has been suggested as a useful mammalian model for a variety of diseases and infections, including infection with respiratory viruses such as SARS-CoV-2. The MesAur1.0 genome assembly was published in 2013 using whole-genome shotgun sequencing with short-read sequence data. Current more advanced sequencing technologies and assembly methods now permit the generation of near-complete genome assemblies with higher quality and higher continuity. Findings Here, we report an improved assembly of the M. auratus genome (BCM_Maur_2.0) using Oxford Nanopore Technologies long-read sequencing to produce a chromosome-scale assembly. The total length of the new assembly is 2.46 Gbp, similar to the 2.50 Gbp length of a previous assembly of this genome, MesAur1.0. BCM_Maur_2.0 exhibits significantly improved continuity with a scaffold N50 that is 6.7 times greater than MesAur1.0. Furthermore, 21,616 protein coding genes and 10,459 noncoding genes were annotated in BCM_Maur_2.0 compared to 20,495 protein coding genes and 4,168 noncoding genes in MesAur1.0. This new assembly also improves the unresolved regions as measured by nucleotide ambiguities, where approximately 17.11% of bases in MesAur1.0 were unresolved compared to BCM_Maur_2.0 in which the number of unresolved bases is reduced to 3.00%. Conclusions Access to a more complete reference genome with improved accuracy and continuity will facilitate more detailed, comprehensive, and meaningful research results for a wide variety of future studies using Syrian hamsters as models.

scholarly journals Chromosome-scale genome assembly of Fusarium oxysporum strain Fo47, a fungal endophyte and biocontrol agent

2020 ◽  
Author(s):  
Bo Wang ◽  
Houlin Yu ◽  
Yanyan Jia ◽  
Quanbin Dong ◽  
Christian Steinberg ◽  
...  
Keyword(s):  
Fusarium Oxysporum ◽  
Genome Assembly ◽  
Plant Disease ◽  
Biocontrol Agent ◽  
Reference Genome ◽  
Fungal Endophyte ◽  
Vascular Wilt ◽  
Sequencing Data ◽  
Plant Disease Control ◽  
Long Read

AbstractHere, we report a chromosome-level genome assembly of Fusarium oxysporum strain Fo47 (12 pseudomolecules; contig N50: 4.52Mb), generated using a combination of PacBio long-read, Illumina pair-ended and Hi-C sequencing data. Although F. oxysporum causes vascular wilt to over 100 plant species, the strain Fo47 is classified as an endophyte and widely used as a biocontrol agent for plant disease control. The Fo47 genome carries a single accessory chromosome of 4.23 Mb, compared to the reference genome of F. oxysporum f.sp. lycopersici strain Fol4287. The high-quality assembly and annotation of the Fo47 genome will be a valuable resource for studying the mechanisms underlying the endophytic interactions between F. oxysporum and plants, as well as deciphering the genome evolution of the F. oxysporum species complex.

scholarly journals Long-Read Genome Assembly of Saccharomyces uvarum Strain CBS 7001

2022 ◽  
Author(s):  
Jingxuan Chen ◽  
David J. Garfinkel ◽  
Casey M. Bergman
Keyword(s):  
Genome Assembly ◽  
Reference Genome ◽  
Sequence Data ◽  
Sensu Stricto ◽  
Whole Genome ◽  
Saccharomyces Uvarum ◽  
Content Type ◽  
Whole Genome Shotgun Sequence ◽  
Long Read ◽  
Genome Shotgun Sequence

Here, we report a long-read genome assembly for Saccharomyces uvarum strain CBS 7001 based on PacBio whole-genome shotgun sequence data. Our assembly provides an improved reference genome for an important yeast in the Saccharomyces sensu stricto clade.

scholarly journals Long read sequencing reveals poxvirus evolution through rapid homogenization of gene arrays

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Thomas A Sasani ◽  
Kelsey R Cone ◽  
Aaron R Quinlan ◽  
Nels C Elde
Keyword(s):  
Copy Number ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Single Nucleotide Variants ◽  
Sequencing Technologies ◽  
Gene Copy Number Variation ◽  
Large Gene ◽  
Oxford Nanopore ◽  
Long Read ◽  
Number Variation

Poxvirus adaptation can involve combinations of recombination-driven gene copy number variation and beneficial single nucleotide variants (SNVs) at the same loci. How these distinct mechanisms of genetic diversification might simultaneously facilitate adaptation to host immune defenses is unknown. We performed experimental evolution with vaccinia virus populations harboring a SNV in a gene actively undergoing copy number amplification. Using long sequencing reads from the Oxford Nanopore Technologies platform, we phased SNVs within large gene copy arrays for the first time. Our analysis uncovered a mechanism of adaptive SNV homogenization reminiscent of gene conversion, which is actively driven by selection. This study reveals a new mechanism for the fluid gain of beneficial mutations in genetic regions undergoing active recombination in viruses and illustrates the value of long read sequencing technologies for investigating complex genome dynamics in diverse biological systems.

scholarly journals Improved draft reference genome for the Glassy-winged Sharpshooter (Homalodisca vitripennis), a vector for Pierce's disease

2021 ◽  
Author(s):  
Cassandra L Ettinger ◽  
Frank J Byrne ◽  
Matthew A Collin ◽  
Derreck Carter-House ◽  
Linda L Walling ◽  
...  
Keyword(s):  
Candidate Genes ◽  
Genome Assembly ◽  
Reference Genome ◽  
Whole Genome Sequence ◽  
Pierce's Disease ◽  
Future Research ◽  
Marker Genes ◽  
Trna Genes ◽  
Homalodisca Vitripennis ◽  
Pierce’S Disease

Homalodisca vitripennis (Hemiptera: Cicadellidae), known as the glassy-winged sharpshooter, is a xylem feeding leafhopper and an important agricultural pest as a vector of Xylella fastidiosa, which causes Pierce's disease in grapes and a variety of other scorch diseases. The current H. vitripennis reference genome from the Baylor College of Medicine's i5k pilot project is a 1.4-Gb assembly with 110,000 scaffolds, which still has significant gaps making identification of genes difficult. To improve on this effort, we used a combination of Oxford Nanopore long-read sequencing technology combined with Illumina sequencing reads to generate a better assembly and first-pass annotation of the whole genome sequence of a wild-caught Californian (Tulare County) individual of H. vitripennis. The improved reference genome assembly for H. vitripennis is 1.93 Gb in length (21,254 scaffolds, N50 = 650 Mb, BUSCO completeness = 94.3%), with 33.06% of the genome masked as repetitive. In total, 108,762 gene models were predicted including 98,296 protein-coding genes and 10,466 tRNA genes. As an additional community resource, we identified 27 orthologous candidate genes of interest for future experimental work including phenotypic marker genes like white. Further, as part of the assembly process, we generated four endosymbiont metagenome-assembled genomes (MAGs), including a high-quality near complete 1.7-Mb Wolbachia sp. genome (1 scaffold, CheckM completeness = 99.4%). The improved genome assembly and annotation for H. vitripennis, curated set of candidate genes, and endosymbiont MAGs will be invaluable resources for future research of H. vitripennis.

42 An Improved, High-quality Ovine Reference Genome Assembly

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 23-24
Author(s):  
Kimberly M Davenport ◽  
Derek M Bickhart ◽  
Kim Worley ◽  
Shwetha C Murali ◽  
Noelle Cockett ◽  
...  
Keyword(s):  
Genome Assembly ◽  
Functional Annotation ◽  
Reference Genome ◽  
De Novo ◽  
The United States ◽  
Read Length ◽  
Chromosome 11 ◽  
High Quality ◽  
Oxford Nanopore ◽  
Long Read

Abstract Sheep are an important agricultural species used for both food and fiber in the United States and globally. A high-quality reference genome enhances the ability to discover genetic and biological mechanisms influencing important traits, such as meat and wool quality. The rapid advances in genome assembly algorithms and emergence of increasingly long sequence read length provide the opportunity for an improved de novo assembly of the sheep reference genome. Tissue was collected postmortem from an adult Rambouillet ewe selected by USDA-ARS for the Ovine Functional Annotation of Animal Genomes project. Short-read (55x coverage), long-read PacBio (75x coverage), and Hi-C data from this ewe were retrieved from public databases. We generated an additional 50x coverage of Oxford Nanopore data and assembled the combined long-read data with canu v1.9. The assembled contigs were polished with Nanopolish v0.12.5 and scaffolded using Hi-C data with Salsa v2.2. Gaps were filled with PBsuite v15.8.24 and polished with Nanopolish v0.12.5 followed by removal of duplicate contigs with PurgeDups v1.0.1. Chromosomes were oriented by identifying centromeres and telomeres with RepeatMasker v4.1.1, indicating a need to reverse the orientation of chromosome 11 relative to Oar_rambouillet_v1.0. Final polishing was performed with two rounds of a pipeline which consisted of freebayes v1.3.1 to call variants, Merfin to validate them, and BCFtools to generate the consensus fasta. The ARS-UI_Ramb_v2.0 assembly has improved continuity (contig N50 of 43.19 Mb) with a 19-fold and 38-fold decrease in the number of scaffolds compared with Oar_rambouillet_v1.0 and Oar_v4.0. ARS-UI_Ramb_v2.0 has greater per-base accuracy and fewer insertions and deletions identified from mapped RNA sequence than previous assemblies. This significantly improved reference assembly, public at NCBI GenBank under accession number GCA_016772045, will optimize the functional annotation of the sheep genome and facilitate improved mapping accuracy of genetic variant and expression data for traits relevant the sheep industry.

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