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April 21, 2020

A full-length transcriptome of Sepia esculenta using a combination of single-molecule long-read (SMRT) and Illumina sequencing

As an economically important cephalopods species, wild-caught Sepia esculenta fishery has suffered a server decline due to over-fishing and ocean environmental damage. To restore this seriously declining fishery resource, we should understand the genetic foundation and molecular mechanism of spawning, reproduction and mortal of golden cuttlefish. In this study, we generated the full-length transcriptome of S. esculenta based on the total RNA of tissue samples (brain, optic gland, nidamental gland, ovary and muscle at different developmental stages) using a combination of single-molecule real-time (SMRT) and Illumina RNA-seq technology. A total of 14.16 Gb SMRT sequencing data were assembled into 94,635 transcripts. Meanwhile, 35.15 Gb Illumina HiSeq data were assembled into 177,226 non-redundant transcripts. Then, we merged SMRT and Illumina assembled data to generate a more complete/full-length S. esculenta transcriptome with 177,951 high-quality transcripts. Based on the obtained transcriptome data, total 81,459 transcripts were annotated in at least one of seven functional databases and 49,189 nucleotide sequences of coding regions were identified. Additionally, 161,327 SSRs distributed in 64,933 transcripts were identified based on SSR analysis. This full-length and high-quality transcriptome of S. esculenta can provide an important foundation for future genomic research on growth and development, reproduction and mortal of cephalopod and further recovery of this recessionary fisheries resources.

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April 21, 2020

Transmission of ESBL-producing Escherichia coli between broilers and humans on broiler farms.

ESBL and AmpC ß-lactamases are an increasing concern for public health. Studies suggest that ESBL/pAmpC-producing Escherichia coli and their plasmids carrying antibiotic resistance genes can spread from broilers to humans working or living on broiler farms. These studies used traditional typing methods, which may not have provided sufficient resolution to reliably assess the relatedness of these isolates.Eleven suspected transmission events among broilers and humans living/working on eight broiler farms were investigated using whole-genome short-read (Illumina) and long-read sequencing (PacBio). Core genome MLST (cgMLST) was performed to investigate the occurrence of strain transmission. Horizontal plasmid and gene transfer were analysed using BLAST.Of eight suspected strain transmission events, six were confirmed. The isolate pairs had identical ESBL/AmpC genes and fewer than eight allelic differences according to the cgMLST, and five had an almost identical plasmid composition. On one of the farms, cgMLST revealed that the isolate pairs belonging to ST10 from a broiler and a household member of the farmer had 475 different alleles, but that the plasmids were identical, indicating horizontal transfer of mobile elements rather than strain transfer. Of three suspected horizontal plasmid transmission events, one was confirmed. In addition, gene transfer between plasmids was found.The present study confirms transmission of strains as well as horizontal plasmid and gene transfer between broilers and farmers and household members on the same farm. WGS is an important tool to confirm suspected zoonotic strain and resistance gene transmission. © The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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April 21, 2020

Assembly of long, error-prone reads using repeat graphs.

Accurate genome assembly is hampered by repetitive regions. Although long single molecule sequencing reads are better able to resolve genomic repeats than short-read data, most long-read assembly algorithms do not provide the repeat characterization necessary for producing optimal assemblies. Here, we present Flye, a long-read assembly algorithm that generates arbitrary paths in an unknown repeat graph, called disjointigs, and constructs an accurate repeat graph from these error-riddled disjointigs. We benchmark Flye against five state-of-the-art assemblers and show that it generates better or comparable assemblies, while being an order of magnitude faster. Flye nearly doubled the contiguity of the human genome assembly (as measured by the NGA50 assembly quality metric) compared with existing assemblers.

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April 21, 2020

Genome sequence of Jatropha curcas L., a non-edible biodiesel plant, provides a resource to improve seed-related traits.

Jatropha curcas (physic nut), a non-edible oilseed crop, represents one of the most promising alternative energy sources due to its high seed oil content, rapid growth and adaptability to various environments. We report ~339 Mbp draft whole genome sequence of J. curcas var. Chai Nat using both the PacBio and Illumina sequencing platforms. We identified and categorized differentially expressed genes related to biosynthesis of lipid and toxic compound among four stages of seed development. Triacylglycerol (TAG), the major component of seed storage oil, is mainly synthesized by phospholipid:diacylglycerol acyltransferase in Jatropha, and continuous high expression of homologs of oleosin over seed development contributes to accumulation of high level of oil in kernels by preventing the breakdown of TAG. A physical cluster of genes for diterpenoid biosynthetic enzymes, including casbene synthases highly responsible for a toxic compound, phorbol ester, in seed cake, was syntenically highly conserved between Jatropha and castor bean. Transcriptomic analysis of female and male flowers revealed the up-regulation of a dozen family of TFs in female flower. Additionally, we constructed a robust species tree enabling estimation of divergence times among nine Jatropha species and five commercial crops in Malpighiales order. Our results will help researchers and breeders increase energy efficiency of this important oil seed crop by improving yield and oil content, and eliminating toxic compound in seed cake for animal feed. © 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

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April 21, 2020

Newly designed 16S rRNA metabarcoding primers amplify diverse and novel archaeal taxa from the environment.

High-throughput studies of microbial communities suggest that Archaea are a widespread component of microbial diversity in various ecosystems. However, proper quantification of archaeal diversity and community ecology remains limited, as sequence coverage of Archaea is usually low owing to the inability of available prokaryotic primers to efficiently amplify archaeal compared to bacterial rRNA genes. To improve identification and quantification of Archaea, we designed and validated the utility of several primer pairs to efficiently amplify archaeal 16S rRNA genes based on up-to-date reference genes. We demonstrate that several of these primer pairs amplify phylogenetically diverse Archaea with high sequencing coverage, outperforming commonly used primers. Based on comparing the resulting long 16S rRNA gene fragments with public databases from all habitats, we found several novel family- to phylum-level archaeal taxa from topsoil and surface water. Our results suggest that archaeal diversity has been largely overlooked due to the limitations of available primers, and that improved primer pairs enable to estimate archaeal diversity more accurately. © 2018 The Authors. Environmental Microbiology Reports published by Society for Applied Microbiology and John Wiley & Sons Ltd.

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April 21, 2020

Genomic variation and strain-specific functional adaptation in the human gut microbiome during early life.

The human gut microbiome matures towards the adult composition during the first years of life and is implicated in early immune development. Here, we investigate the effects of microbial genomic diversity on gut microbiome development using integrated early childhood data sets collected in the DIABIMMUNE study in Finland, Estonia and Russian Karelia. We show that gut microbial diversity is associated with household location and linear growth of children. Single nucleotide polymorphism- and metagenomic assembly-based strain tracking revealed large and highly dynamic microbial pangenomes, especially in the genus Bacteroides, in which we identified evidence of variability deriving from Bacteroides-targeting bacteriophages. Our analyses revealed functional consequences of strain diversity; only 10% of Finnish infants harboured Bifidobacterium longum subsp. infantis, a subspecies specialized in human milk metabolism, whereas Russian infants commonly maintained a probiotic Bifidobacterium bifidum strain in infancy. Groups of bacteria contributing to diverse, characterized metabolic pathways converged to highly subject-specific configurations over the first two years of life. This longitudinal study extends the current view of early gut microbial community assembly based on strain-level genomic variation.

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April 21, 2020

Antarctic blackfin icefish genome reveals adaptations to extreme environments.

Icefishes (suborder Notothenioidei; family Channichthyidae) are the only vertebrates that lack functional haemoglobin genes and red blood cells. Here, we report a high-quality genome assembly and linkage map for the Antarctic blackfin icefish Chaenocephalus aceratus, highlighting evolved genomic features for its unique physiology. Phylogenomic analysis revealed that Antarctic fish of the teleost suborder Notothenioidei, including icefishes, diverged from the stickleback lineage about 77 million years ago and subsequently evolved cold-adapted phenotypes as the Southern Ocean cooled to sub-zero temperatures. Our results show that genes involved in protection from ice damage, including genes encoding antifreeze glycoprotein and zona pellucida proteins, are highly expanded in the icefish genome. Furthermore, genes that encode enzymes that help to control cellular redox state, including members of the sod3 and nqo1 gene families, are expanded, probably as evolutionary adaptations to the relatively high concentration of oxygen dissolved in cold Antarctic waters. In contrast, some crucial regulators of circadian homeostasis (cry and per genes) are absent from the icefish genome, suggesting compromised control of biological rhythms in the polar light environment. The availability of the icefish genome sequence will accelerate our understanding of adaptation to extreme Antarctic environments.

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April 21, 2020

Genome sequence and genetic transformation of a widely distributed and cultivated poplar.

Populus alba is widely distributed and cultivated in Europe and Asia. This species has been used for diverse studies. In this study, we assembled a de novo genome sequence of P. alba var. pyramidalis (= P. bolleana) and confirmed its high transformation efficiency and short transformation time by experiments. Through a process of hybrid genome assembly, a total of 464 M of the genome was assembled. Annotation analyses predicted 37 901 protein-coding genes. This genome is highly collinear to that of P. trichocarpa, with most genes having orthologs in the two species. We found a marked expansion of gene families related to histone and the hormone auxin but loss of disease resistance genes in P. alba if compared with the closely related P. trichocarpa. The genome sequence presented here represents a valuable resource for further molecular functional analyses of this species as a new tree model, poplar breeding practices and comparative genomic analyses across different poplars. © 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

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April 21, 2020

Patterns of non-ARD variation in more than 300 full-length HLA-DPB1 alleles.

Our understanding of sequence variation in the HLA-DPB1 gene is largely restricted to the hypervariable antigen recognition domain (ARD) encoded by exon 2. Here, we employed a redundant sequencing strategy combining long-read and short-read data to accurately phase and characterise in full length the majority of common and well-documented (CWD) DPB1 alleles as well as alleles with an observed frequency of at least 0.0006% in our predominantly European sample set. We generated 664 DPB1 sequences, comprising 279 distinct allelic variants. This allows us to present the, to date, most comprehensive analysis of the nature and extent of DPB1 sequence variation. The full-length sequence analysis revealed the existence of two highly diverged allele clades. These clades correlate with the rs9277534 A???G variant, a known expression marker located in the 3′-UTR. The two clades are fully differentiated by 174 fixed polymorphisms throughout a 3.6?kb stretch at the 3′-end of DPB1. The region upstream of this differentiation zone is characterised by increasingly shared variation between the clades. The low-expression A clade comprises 59% of the distinct allelic sequences including the three by far most frequent DPB1 alleles, DPB1*04:01, DPB1*02:01 and DPB1*04:02. Alleles in the A clade show reduced nucleotide diversity with an excess of rare variants when compared to the high-expression G clade. This pattern is consistent with a scenario of recent proliferation of A-clade alleles. The full-length characterisation of all but the most rare DPB1 alleles will benefit the application of NGS for DPB1 genotyping and provides a helpful framework for a deeper understanding of high- and low-expression alleles and their implications in the context of unrelated haematopoietic stem-cell transplantation.Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

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April 21, 2020

Efficiency of PacBio long read correction by 2nd generation Illumina sequencing.

Long sequencing reads offer unprecedented opportunities in analysis and reconstruction of complex genomic regions. However, the gain in sequence length is often traded for quality. Therefore, recently several approaches have been proposed (e.g. higher sequencing coverage, hybrid assembly or sequence correction) to enhance the quality of long sequencing reads. A simple and cost-effective approach includes use of the high quality 2nd generation sequencing data to improve the quality of long reads. We designed a dedicated testing procedure and selected universal programs for long read correction, which provide as the output sequences that can be used in further genomic and transcriptomic studies. Our results show that HALC is the best choice for correction of long PacBio reads, when both, read size and quality, are the main focus of the analysis. However, the tested tools show some unexpected behaviors, including read trimming and fragmentation.Copyright © 2017 Elsevier Inc. All rights reserved.

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April 21, 2020

Genetic map-guided genome assembly reveals a virulence-governing minichromosome in the lentil anthracnose pathogen Colletotrichum lentis.

Colletotrichum lentis causes anthracnose, which is a serious disease on lentil and can account for up to 70% crop loss. Two pathogenic races, 0 and 1, have been described in the C. lentis population from lentil. To unravel the genetic control of virulence, an isolate of the virulent race 0 was sequenced at 1481-fold genomic coverage. The 56.10-Mb genome assembly consists of 50 scaffolds with N50 scaffold length of 4.89 Mb. A total of 11 436 protein-coding gene models was predicted in the genome with 237 coding candidate effectors, 43 secondary metabolite biosynthetic enzymes and 229 carbohydrate-active enzymes (CAZymes), suggesting a contraction of the virulence gene repertoire in C. lentis. Scaffolds were assigned to 10 core and two minichromosomes using a population (race 0 × race 1, n = 94 progeny isolates) sequencing-based, high-density (14 312 single nucleotide polymorphisms) genetic map. Composite interval mapping revealed a single quantitative trait locus (QTL), qClVIR-11, located on minichromosome 11, explaining 85% of the variability in virulence of the C. lentis population. The QTL covers a physical distance of 0.84 Mb with 98 genes, including seven candidate effector and two secondary metabolite genes. Taken together, the study provides genetic and physical evidence for the existence of a minichromosome controlling the C. lentis virulence on lentil. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

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April 21, 2020

An Annotated Genome for Haliotis rufescens (Red Abalone) and Resequenced Green, Pink, Pinto, Black, and White Abalone Species.

Abalone are one of the few marine taxa where aquaculture production dominates the global market as a result of increasing demand and declining natural stocks from overexploitation and disease. To better understand abalone biology, aid in conservation efforts for endangered abalone species, and gain insight into sustainable aquaculture, we created a draft genome of the red abalone (Haliotis rufescens). The approach to this genome draft included initial assembly using raw Illumina and PacBio sequencing data with MaSuRCA, before scaffolding using sequencing data generated from Chicago library preparations with HiRise2. This assembly approach resulted in 8,371 scaffolds and total length of 1.498?Gb; the N50 was 1.895?Mb, and the longest scaffold was 13.2?Mb. Gene models were predicted, using MAKER2, from RNA-Seq data and all related expressed sequence tags and proteins from NCBI; this resulted in 57,785 genes with an average length of 8,255?bp. In addition, single nucleotide polymorphisms were called on Illumina short-sequencing reads from five other eastern Pacific abalone species: the green (H. fulgens), pink (H. corrugata), pinto (H. kamtschatkana), black (H. cracherodii), and white (H. sorenseni) abalone. Phylogenetic relationships largely follow patterns detected by previous studies based on 1,784,991 high-quality single nucleotide polymorphisms. Among the six abalone species examined, the endangered white abalone appears to harbor the lowest levels of heterozygosity. This draft genome assembly and the sequencing data provide a foundation for genome-enabled aquaculture improvement for red abalone, and for genome-guided conservation efforts for the other five species and, in particular, for the endangered white and black abalone.

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April 21, 2020

In-depth analysis of the genome of Trypanosoma evansi, an etiologic agent of surra.

Trypanosoma evansi is the causative agent of the animal trypanosomiasis surra, a disease with serious economic burden worldwide. The availability of the genome of its closely related parasite Trypanosoma brucei allows us to compare their genetic and evolutionarily shared and distinct biological features. The complete genomic sequence of the T. evansi YNB strain was obtained using a combination of genomic and transcriptomic sequencing, de novo assembly, and bioinformatic analysis. The genome size of the T. evansi YNB strain was 35.2 Mb, showing 96.59% similarity in sequence and 88.97% in scaffold alignment with T. brucei. A total of 8,617 protein-coding genes, accounting for 31% of the genome, were predicted. Approximately 1,641 alternative splicing events of 820 genes were identified, with a majority mediated by intron retention, which represented a major difference in post-transcriptional regulation between T. evansi and T. brucei. Disparities in gene copy number of the variant surface glycoprotein, expression site-associated genes, microRNAs, and RNA-binding protein were clearly observed between the two parasites. The results revealed the genomic determinants of T. evansi, which encoded specific biological characteristics that distinguished them from other related trypanosome species.

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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 are related to sex floral development. This 59-Kb female-specific region was likely to be derived from duplication and rearrangement of paralogous genes and retained non-recombinant in the female-specific region. Sex-specific molecular markers developed from candidate genes co-segregated with sex in a genetically diverse female and male germplasm. We propose sex determination follow the ZW model of female heterogamety. The genome sequence of red bayberry provides a valuable resource for plant sex chromosome evolution and also provides important insights for molecular biology, genetics and modern breeding in Myricaceae family. © 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

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April 21, 2020

Discovery of tandem and interspersed segmental duplications using high-throughput sequencing.

Several algorithms have been developed that use high-throughput sequencing technology to characterize structural variations (SVs). Most of the existing approaches focus on detecting relatively simple types of SVs such as insertions, deletions and short inversions. In fact, complex SVs are of crucial importance and several have been associated with genomic disorders. To better understand the contribution of complex SVs to human disease, we need new algorithms to accurately discover and genotype such variants. Additionally, due to similar sequencing signatures, inverted duplications or gene conversion events that include inverted segmental duplications are often characterized as simple inversions, likewise, duplications and gene conversions in direct orientation may be called as simple deletions. Therefore, there is still a need for accurate algorithms to fully characterize complex SVs and thus improve calling accuracy of more simple variants.We developed novel algorithms to accurately characterize tandem, direct and inverted interspersed segmental duplications using short read whole genome sequencing datasets. We integrated these methods to our TARDIS tool, which is now capable of detecting various types of SVs using multiple sequence signatures such as read pair, read depth and split read. We evaluated the prediction performance of our algorithms through several experiments using both simulated and real datasets. In the simulation experiments, using a 30× coverage TARDIS achieved 96% sensitivity with only 4% false discovery rate. For experiments that involve real data, we used two haploid genomes (CHM1 and CHM13) and one human genome (NA12878) from the Illumina Platinum Genomes set. Comparison of our results with orthogonal PacBio call sets from the same genomes revealed higher accuracy for TARDIS than state-of-the-art methods. Furthermore, we showed a surprisingly low false discovery rate of our approach for discovery of tandem, direct and inverted interspersed segmental duplications prediction on CHM1 (<5% for the top 50 predictions).TARDIS source code is available at https://github.com/BilkentCompGen/tardis, and a corresponding Docker image is available at https://hub.docker.com/r/alkanlab/tardis/.Supplementary data are available at Bioinformatics online. © The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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