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Sunday, October 25, 2020

i5K Webinar: High-quality de novo insect genome assemblies using PacBio sequencing

PacBio Sequencing is characterized by very long sequence reads (averaging > 10,000 bases), lack of GC-bias, and high consensus accuracy. These features have allowed the method to provide a new gold standard in de novo genome assemblies, producing highly contiguous (contig N50 > 1 Mb) and accurate (> QV 50) genome assemblies. We will briefly describe the technology and then highlight the full workflow, from sample preparation through sequencing to data analysis, on examples of insect genome assemblies, and illustrate the difference these high-quality genomes represent with regard to biological insights, compared to fragmented draft assemblies generated by short-read sequencing.

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Tuesday, April 21, 2020

De novo genome assembly of the endangered Acer yangbiense, a plant species with extremely small populations endemic to Yunnan Province, China.

Acer yangbiense is a newly described critically endangered endemic maple tree confined to Yangbi County in Yunnan Province in Southwest China. It was included in a programme for rescuing the most threatened species in China, focusing on “plant species with extremely small populations (PSESP)”.We generated 64, 94, and 110 Gb of raw DNA sequences and obtained a chromosome-level genome assembly of A. yangbiense through a combination of Pacific Biosciences Single-molecule Real-time, Illumina HiSeq X, and Hi-C mapping, respectively. The final genome assembly is ~666 Mb, with 13 chromosomes covering ~97% of the genome and scaffold N50 sizes of 45 Mb.…

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Tuesday, April 21, 2020

The genomes of pecan and Chinese hickory provide insights into Carya evolution and nut nutrition.

Pecan (Carya illinoinensis) and Chinese hickory (C. cathayensis) are important commercially cultivated nut trees in the genus Carya (Juglandaceae), with high nutritional value and substantial health benefits.We obtained >187.22 and 178.87 gigabases of sequence, and ~288× and 248× genome coverage, to a pecan cultivar (“Pawnee”) and a domesticated Chinese hickory landrace (ZAFU-1), respectively. The total assembly size is 651.31 megabases (Mb) for pecan and 706.43 Mb for Chinese hickory. Two genome duplication events before the divergence from walnut were found in these species. Gene family analysis highlighted key genes in biotic and abiotic tolerance, oil, polyphenols, essential amino acids, and…

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Tuesday, April 21, 2020

Comprehensive transcriptome analysis reveals genes potentially involved in isoflavone biosynthesis in Pueraria thomsonii Benth.

Pueraria thomsonii Benth is an important medicinal plant. Transcriptome sequencing, unigene assembly, the annotation of transcripts and the study of gene expression profiles play vital roles in gene function research. However, the full-length transcriptome of P. thomsonii remains unknown. Here, we obtained 44,339 nonredundant transcripts of P. thomsonii by using the PacBio RS II Isoform and Illumina sequencing platforms, of which 43,195 were annotated genes. Compared with the expression levels in the plant roots, those of transcripts with a |fold change| = 4 and FDR < 0.01 in the leaves or stems were assigned as differentially expressed transcripts (DETs). In…

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Tuesday, April 21, 2020

µLAS technology for DNA isolation coupled to Cas9-assisted targeting for sequencing and assembly of a 30 kb region in plant genome.

Cas9-assisted targeting of DNA fragments in complex genomes is viewed as an essential strategy to obtain high-quality and continuous sequence data. However, the purity of target loci selected by pulsed-field gel electrophoresis (PFGE) has so far been insufficient to assemble the sequence in one contig. Here, we describe the µLAS technology to capture and purify high molecular weight DNA. First, the technology is optimized to perform high sensitivity DNA profiling with a limit of detection of 20 fg/µl for 50 kb fragments and an analytical time of 50 min. Then, µLAS is operated to isolate a 31.5 kb locus cleaved…

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Tuesday, April 21, 2020

Tools and Strategies for Long-Read Sequencing and De Novo Assembly of Plant Genomes.

The commercial release of third-generation sequencing technologies (TGSTs), giving long and ultra-long sequencing reads, has stimulated the development of new tools for assembling highly contiguous genome sequences with unprecedented accuracy across complex repeat regions. We survey here a wide range of emerging sequencing platforms and analytical tools for de novo assembly, provide background information for each of their steps, and discuss the spectrum of available options. Our decision tree recommends workflows for the generation of a high-quality genome assembly when used in combination with the specific needs and resources of a project.Copyright © 2019 Elsevier Ltd. All rights reserved.

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Tuesday, April 21, 2020

De novo assembly of white poplar genome and genetic diversity of white poplar population in Irtysh River basin in China.

The white poplar (Populus alba) is widely distributed in Central Asia and Europe. There are natural populations of white poplar in Irtysh River basin in China. It also can be cultivated and grown well in northern China. In this study, we sequenced the genome of P. alba by single-molecule real-time technology. De novo assembly of P. alba had a genome size of 415.99 Mb with a contig N50 of 1.18 Mb. A total of 32,963 protein-coding genes were identified. 45.16% of the genome was annotated as repetitive elements. Genome evolution analysis revealed that divergence between P. alba and Populus trichocarpa…

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Tuesday, April 21, 2020

Diversity of phytobeneficial traits revealed by whole-genome analysis of worldwide-isolated phenazine-producing Pseudomonas spp.

Plant-beneficial Pseudomonas spp. competitively colonize the rhizosphere and display plant-growth promotion and/or disease-suppression activities. Some strains within the P. fluorescens species complex produce phenazine derivatives, such as phenazine-1-carboxylic acid. These antimicrobial compounds are broadly inhibitory to numerous soil-dwelling plant pathogens and play a role in the ecological competence of phenazine-producing Pseudomonas spp. We assembled a collection encompassing 63 strains representative of the worldwide diversity of plant-beneficial phenazine-producing Pseudomonas spp. In this study, we report the sequencing of 58 complete genomes using PacBio RS II sequencing technology. Distributed among four subgroups within the P. fluorescens species complex, the diversity of our…

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Tuesday, April 21, 2020

The red bayberry genome and genetic basis of sex determination.

Morella rubra, red bayberry, is an economically important fruit tree in south China. Here, we assembled the first high-quality genome for both a female and a male individual of red bayberry. The genome size was 313-Mb, and 90% sequences were assembled into eight pseudo chromosome molecules, with 32 493 predicted genes. By whole-genome comparison between the female and male and association analysis with sequences of bulked and individual DNA samples from female and male, a 59-Kb region determining female was identified and located on distal end of pseudochromosome 8, which contains abundant transposable element and seven putative genes, four of them…

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Tuesday, April 21, 2020

A reference genome for pea provides insight into legume genome evolution.

We report the first annotated chromosome-level reference genome assembly for pea, Gregor Mendel’s original genetic model. Phylogenetics and paleogenomics show genomic rearrangements across legumes and suggest a major role for repetitive elements in pea genome evolution. Compared to other sequenced Leguminosae genomes, the pea genome shows intense gene dynamics, most likely associated with genome size expansion when the Fabeae diverged from its sister tribes. During Pisum evolution, translocation and transposition differentially occurred across lineages. This reference sequence will accelerate our understanding of the molecular basis of agronomically important traits and support crop improvement.

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Tuesday, April 21, 2020

The role of genomic structural variation in the genetic improvement of polyploid crops

Many of our major crop species are polyploids, containing more than one genome or set of chromosomes. Polyploid crops present unique challenges, including difficulties in genome assembly, in discriminating between multiple gene and sequence copies, and in genetic mapping, hindering use of genomic data for genetics and breeding. Polyploid genomes may also be more prone to containing structural variation, such as loss of gene copies or sequences (presence–absence variation) and the presence of genes or sequences in multiple copies (copy-number variation). Although the two main types of genomic structural variation commonly identified are presence–absence variation and copy-number variation, we propose…

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Tuesday, April 21, 2020

A reference-grade wild soybean genome.

Efficient crop improvement depends on the application of accurate genetic information contained in diverse germplasm resources. Here we report a reference-grade genome of wild soybean accession W05, with a final assembled genome size of 1013.2?Mb and a contig N50 of 3.3?Mb. The analytical power of the W05 genome is demonstrated by several examples. First, we identify an inversion at the locus determining seed coat color during domestication. Second, a translocation event between chromosomes 11 and 13 of some genotypes is shown to interfere with the assignment of QTLs. Third, we find a region containing copy number variations of the Kunitz…

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Tuesday, April 21, 2020

Chromosome-level assembly of the water buffalo genome surpasses human and goat genomes in sequence contiguity.

Rapid innovation in sequencing technologies and improvement in assembly algorithms have enabled the creation of highly contiguous mammalian genomes. Here we report a chromosome-level assembly of the water buffalo (Bubalus bubalis) genome using single-molecule sequencing and chromatin conformation capture data. PacBio Sequel reads, with a mean length of 11.5?kb, helped to resolve repetitive elements and generate sequence contiguity. All five B. bubalis sub-metacentric chromosomes were correctly scaffolded with centromeres spanned. Although the index animal was partly inbred, 58% of the genome was haplotype-phased by FALCON-Unzip. This new reference genome improves the contig N50 of the previous short-read based buffalo assembly…

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Tuesday, April 21, 2020

Full-length transcript sequencing and comparative transcriptomic analysis to evaluate the contribution of osmotic and ionic stress components towards salinity tolerance in the roots of cultivated alfalfa (Medicago sativa L.).

Alfalfa is the most extensively cultivated forage legume. Salinity is a major environmental factor that impacts on alfalfa’s productivity. However, little is known about the molecular mechanisms underlying alfalfa responses to salinity, especially the relative contribution of the two important components of osmotic and ionic stress.In this study, we constructed the first full-length transcriptome database for alfalfa root tips under continuous NaCl and mannitol treatments for 1, 3, 6, 12, and 24?h (three biological replicates for each time points, including the control group) via PacBio Iso-Seq. This resulted in the identification of 52,787 full-length transcripts, with an average length of…

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