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July 7, 2019

Genome sequencing and analysis of the first complete genome of Lactobacillus kunkeei strain MP2, an Apis mellifera gut isolate

Background. The honey bee (Apis mellifera) is the most important pollinator in agriculture worldwide. However, the number of honey bees has fallen significantly since 2006, becoming a huge ecological problem nowadays. The principal cause is CCD, or Colony Collapse Disorder, characterized by the seemingly spontaneous abandonment of hives by their workers. One of the characteristics of CCD in honey bees is the alteration of the bacterial communities in their gastrointestinal tract, mainly due to the decrease of Firmicutes populations, such as the Lactobacilli. At this time, the causes of these alterations remain unknown. We recently isolated a strain of Lactobacillus kunkeei (L. kunkeei strain MP2) from the gut of Chilean honey bees. L. kunkeei, is one of the most commonly isolated bacterium from the honey bee gut and is highly versatile in different ecological niches. In this study, we aimed to elucidate in detail, the L. kunkeei genetic background and perform a comparative genome analysis with other Lactobacillus species. Methods. L. kunkeei MP2 was originally isolated from the guts of Chilean A. mellifera individuals. Genome sequencing was done using Pacific Biosciences single-molecule real-time sequencing technology. De novo assembly was performed using Celera assembler. The genome was annotated using Prokka, and functional information was added using the EggNOG 3.1 database. In addition, genomic islands were predicted using IslandViewer, and pro-phage sequences using PHAST. Comparisons between L. kunkeei MP2 with other L. kunkeei, and Lactobacillus strains were done using Roary. Results. The complete genome of L. kunkeei MP2 comprises one circular chromosome of 1,614,522 nt. with a GC content of 36,9%. Pangenome analysis with 16 L. kunkeei strains, identified 113 unique genes, most of them related to phage insertions. A large and unique region of L. kunkeei MP2 genome contains several genes that encode for phage structural protein and replication components. Comparative analysis of MP2 with other Lactobacillus species, identified several unique genes of L. kunkeei MP2 related with metabolism, biofilm generation, survival under stress conditions, and mobile genetic elements (MGEs). Discussion. The presence of multiple mobile genetic elements, including phage sequences, suggest a high degree of genetic variability in L. kunkeei. Its versatility and ability to survive in different ecological niches (bee guts, flowers, fruits among others) could be given by its genetic capacity to change and adapt to different environments. L. kunkeei could be a new source of Lactobacillus with beneficial properties. Indeed, L. kunkeei MP2 could play an important role in honey bee nutrition through the synthesis of components as isoprenoids.

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July 7, 2019

Single-molecule sequencing assists genome assembly improvement and structural variation inference.

Dear editor, The single-molecule real-time (SMRT) sequencing platform presented by Pacific Biosciences (PacBio) is regarded as a third-generation sequencing technology (Eid et al., 2009, Roberts et al., 2013). PacBio delivers long reads from several to tens of kilobases (kbs), which are ideal for filling unsequenced gaps due to unusual sequence contexts, such as high-GC content or repeat-rich regions (Bashir et al., 2012, Berlin et al., 2015, Chaisson et al., 2015). PacBio long reads are also favorable for detecting large DNA fragments harboring structural variations (SVs), such as inversions, translocations, duplications, and large insertions/deletions (indels) (Ritz et al., 2010, English et al., 2014). However, one drawback of PacBio is the high error rate of base calling for single pass coverage of the genome (Au et al., 2012, Koren et al., 2012). This drawback can be mitigated by increasing sequencing coverage to achieve high consensus accuracy, but the requirements may be prohibitive for the de novo assembly of large- or medium-size genomes using only PacBio when considering both budgetary and computational costs. Alternatively, PacBio may be used for assembly improvement of near-finished reference genomes, especially for filling gaps in which unsequenced bases are represented by the letter N (English et al., 2012). Here, we combined PacBio (~15x) with Illumina reads (~40x) to improve the genome assemblies of African wild (Oryza barthii) and cultivated rice (O. glaberrima), and to infer large SVs between O. barthii and O. glaberrima.

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July 7, 2019

Evolutionary redesign of the Atlantic cod (Gadus morhua L.) Toll-like receptor repertoire by gene losses and expansions.

Genome sequencing of the teleost Atlantic cod demonstrated loss of the Major Histocompatibility Complex (MHC) class II, an extreme gene expansion of MHC class I and gene expansions and losses in the innate pattern recognition receptor (PRR) family of Toll-like receptors (TLR). In a comparative genomic setting, using an improved version of the genome, we characterize PRRs in Atlantic cod with emphasis on TLRs demonstrating the loss of TLR1/6, TLR2 and TLR5 and expansion of TLR7, TLR8, TLR9, TLR22 and TLR25. We find that Atlantic cod TLR expansions are strongly influenced by diversifying selection likely to increase the detectable ligand repertoire through neo- and subfunctionalization. Using RNAseq we find that Atlantic cod TLRs display likely tissue or developmental stage-specific expression patterns. In a broader perspective, a comprehensive vertebrate TLR phylogeny reveals that the Atlantic cod TLR repertoire is extreme with regards to losses and expansions compared to other teleosts. In addition we identify a substantial shift in TLR repertoires following the evolutionary transition from an aquatic vertebrate (fish) to a terrestrial (tetrapod) life style. Collectively, our findings provide new insight into the function and evolution of TLRs in Atlantic cod as well as the evolutionary history of vertebrate innate immunity.

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July 7, 2019

Variable presence of the inverted repeat and plastome stability in Erodium.

Several unrelated lineages such as plastids, viruses and plasmids, have converged on quadripartite genomes of similar size with large and small single copy regions and a large inverted repeat (IR). Except for Erodium (Geraniaceae), saguaro cactus and some legumes, the plastomes of all photosynthetic angiosperms display this structure. The functional significance of the IR is not understood and Erodium provides a system to examine the role of the IR in the long-term stability of these genomes. We compared the degree of genomic rearrangement in plastomes of Erodium that differ in the presence and absence of the IR.We sequenced 17 new Erodium plastomes. Using 454, Illumina, PacBio and Sanger sequences, 16 genomes were assembled and categorized along with one incomplete and two previously published Erodium plastomes. We conducted phylogenetic analyses among these species using a dataset of 19 protein-coding genes and determined if significantly higher evolutionary rates had caused the long branch seen previously in phylogenetic reconstructions within the genus. Bioinformatic comparisons were also performed to evaluate plastome evolution across the genus.Erodium plastomes fell into four types (Type 1-4) that differ in their substitution rates, short dispersed repeat content and degree of genomic rearrangement, gene and intron content and GC content. Type 4 plastomes had significantly higher rates of synonymous substitutions (dS) for all genes and for 14 of the 19 genes non-synonymous substitutions (dN) were significantly accelerated. We evaluated the evidence for a single IR loss in Erodium and in doing so discovered that Type 4 plastomes contain a novel IR.The presence or absence of the IR does not affect plastome stability in Erodium. Rather, the overall repeat content shows a negative correlation with genome stability, a pattern in agreement with other angiosperm groups and recent findings on genome stability in bacterial endosymbionts.© The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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July 7, 2019

Haemonchus contortus: genome structure, organization and comparative genomics

One of the first genome sequencing projects for a parasitic nematode was that for Haemonchus contortus. The open access data from the Wellcome Trust Sanger Institute provided a valuable early resource for the research community, particularly for the identification of specific genes and genetic markers. Later, a second sequencing project was initiated by the University of Melbourne, and the two draft genome sequences for H. contortus were published back-to-back in 2013. There is a pressing need for long-range genomic information for genetic mapping, population genetics and functional genomic studies, so we are continuing to improve the Wellcome Trust Sanger Institute assembly to provide a finished reference genome for H. contortus. This review describes this process, compares the H. contortus genome assemblies with draft genomes from other members of the strongylid group and discusses future directions for parasite genomics using the H. contortus model. Copyright © 2016 Elsevier Ltd. All rights reserved.

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July 7, 2019

Analysis of the genome sequence of the medicinal plant Salvia miltiorrhiza.

Salvia miltiorrhiza Bunge (Danshen) is a medicinal plant of the Lamiaceae family, and its dried roots have long been used in traditional Chinese medicine with hydrophilic phenolic acids and tanshinones as pharmaceutically active components (Zhang et al., 2014; Xu et al., 2016). The first step of tanshinone biosynthesis is bicyclization of the general diterpene precursor (E,E,E)-geranylgeranyl diphosphate (GGPP) to copalyl diphosphate (CPP) by CPP synthases (CPSs), which is followed by a cyclization or rearrangement reaction catalyzed by kaurene synthase-like enzymes (KSL).

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July 7, 2019

Genome sequence and analysis of a stress-tolerant, wild-derived strain of Saccharomyces cerevisiae used in biofuels research

The genome sequences of more than 100 strains of the yeast Saccharomyces cerevisiae have been published. Unfortunately, most of these genome assemblies contain dozens to hundreds of gaps at repetitive sequences, including transposable elements, tRNAs, and subtelomeric regions, which is where novel genes generally reside. Relatively few strains have been chosen for genome sequencing based on their biofuel production potential, leaving an additional knowledge gap. Here, we describe the nearly complete genome sequence of GLBRCY22-3 (Y22-3), a strain of S. cerevisiae derived from the stress-tolerant wild strain NRRL YB-210 and subsequently engineered for xylose metabolism. After benchmarking several genome assembly approaches, we developed a pipeline to integrate Pacific Biosciences (PacBio) and Illumina sequencing data and achieved one of the highest quality genome assemblies for any S. cerevisiae strain. Specifically, the contig N50 is 693 kbp, and the sequences of most chromosomes, the mitochondrial genome, and the 2-micron plasmid are complete. Our annotation predicts 92 genes that are not present in the reference genome of the laboratory strain S288c, over 70% of which were expressed. We predicted functions for 43 of these genes, 28 of which were previously uncharacterized and unnamed. Remarkably, many of these genes are predicted to be involved in stress tolerance and carbon metabolism and are shared with a Brazilian bioethanol production strain, even though the strains differ dramatically at most genetic loci. The Y22-3 genome sequence provides an exceptionally high-quality resource for basic and applied research in bioenergy and genetics. Copyright © 2016 McIlwain et al.

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July 7, 2019

An improved genome assembly of Azadirachta indica A. Juss.

Neem (Azadirachta indica A. Juss.), an evergreen tree of the Meliaceae family, is known for its medicinal, cosmetic, pesticidal and insecticidal properties. We had previously sequenced and published the draft genome of the plant, using mainly short read sequencing data. In this report, we present an improved genome assembly generated using additional short reads from Illumina and long reads from Pacific Biosciences SMRT sequencer. We assembled short reads and error corrected long reads using Platanus, an assembler designed to perform well for heterozygous genomes. The updated genome assembly (v2.0) yielded 3- and 3.5-fold increase in N50 and N75, respectively; 2.6-fold decrease in the total number of scaffolds; 1.25-fold increase in the number of valid transcriptome alignments; 13.4-fold less mis-assembly and 1.85-fold increase in the percentage repeat, over the earlier assembly (v1.0). The current assembly also maps better to the genes known to be involved in the terpenoid biosynthesis pathway. Together, the data represents an improved assembly of the A. indica genome. The raw data described in this manuscript are submitted to the NCBI Short Read Archive under the accession numbers SRX1074131, SRX1074132, SRX1074133, and SRX1074134 (SRP013453). Copyright © 2016 Author et al.

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July 7, 2019

PEPR: pipelines for evaluating prokaryotic references.

The rapid adoption of microbial whole genome sequencing in public health, clinical testing, and forensic laboratories requires the use of validated measurement processes. Well-characterized, homogeneous, and stable microbial genomic reference materials can be used to evaluate measurement processes, improving confidence in microbial whole genome sequencing results. We have developed a reproducible and transparent bioinformatics tool, PEPR, Pipelines for Evaluating Prokaryotic References, for characterizing the reference genome of prokaryotic genomic materials. PEPR evaluates the quality, purity, and homogeneity of the reference material genome, and purity of the genomic material. The quality of the genome is evaluated using high coverage paired-end sequence data; coverage, paired-end read size and direction, as well as soft-clipping rates, are used to identify mis-assemblies. The homogeneity and purity of the material relative to the reference genome are characterized by comparing base calls from replicate datasets generated using multiple sequencing technologies. Genomic purity of the material is assessed by checking for DNA contaminants. We demonstrate the tool and its output using sequencing data while developing a Staphylococcus aureus candidate genomic reference material. PEPR is open source and available at https://github.com/usnistgov/pepr .

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July 7, 2019

Genome sequence of Ustilaginoidea virens IPU010, a rice pathogenic fungus causing false smut.

Ustilaginoidea virens is a rice pathogenic fungus that causes false smut disease, a disease that seriously damages the yield and quality of the grain. Analysis of the U. virens IPU010 33.6-Mb genome sequence will aid in the understanding of the pathogenicity of the strain, particularly in regard to effector proteins and secondary metabolic genes. Copyright © 2016 Kumagai et al.

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July 7, 2019

Near-Complete Genome Sequence of Clostridium paradoxum Strain JW-YL-7.

Clostridium paradoxum strain JW-YL-7 is a moderately thermophilic anaerobic alkaliphile isolated from the municipal sewage treatment plant in Athens, GA. We report the near-complete genome sequence of C. paradoxum strain JW-YL-7 obtained by using PacBio DNA sequencing and Pilon for sequence assembly refinement with Illumina data. Copyright © 2016 Lancaster et al.

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July 7, 2019

Elucidating the triplicated ancestral genome structure of radish based on chromosome-level comparison with the Brassica genomes.

This study presents a chromosome-scale draft genome sequence of radish that is assembled into nine chromosomal pseudomolecules. A comprehensive comparative genome analysis with the Brassica genomes provides genomic evidences on the evolution of the mesohexaploid radish genome. Radish (Raphanus sativus L.) is an agronomically important root vegetable crop and its origin and phylogenetic position in the tribe Brassiceae is controversial. Here we present a comprehensive analysis of the radish genome based on the chromosome sequences of R. sativus cv. WK10039. The radish genome was sequenced and assembled into 426.2 Mb spanning >98 % of the gene space, of which 344.0 Mb were integrated into nine chromosome pseudomolecules. Approximately 36 % of the genome was repetitive sequences and 46,514 protein-coding genes were predicted and annotated. Comparative mapping of the tPCK-like ancestral genome revealed that the radish genome has intermediate characteristics between the Brassica A/C and B genomes in the triplicated segments, suggesting an internal origin from the genus Brassica. The evolutionary characteristics shared between radish and other Brassica species provided genomic evidences that the current form of nine chromosomes in radish was rearranged from the chromosomes of hexaploid progenitor. Overall, this study provides a chromosome-scale draft genome sequence of radish as well as novel insight into evolution of the mesohexaploid genomes in the tribe Brassiceae.

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July 7, 2019

OPERA-LG: efficient and exact scaffolding of large, repeat-rich eukaryotic genomes with performance guarantees.

The assembly of large, repeat-rich eukaryotic genomes represents a significant challenge in genomics. While long-read technologies have made the high-quality assembly of small, microbial genomes increasingly feasible, data generation can be expensive for larger genomes. OPERA-LG is a scalable, exact algorithm for the scaffold assembly of large, repeat-rich genomes, out-performing state-of-the-art programs for scaffold correctness and contiguity. It provides a rigorous framework for scaffolding of repetitive sequences and a systematic approach for combining data from different second-generation and third-generation sequencing technologies. OPERA-LG provides an avenue for systematic augmentation and improvement of thousands of existing draft eukaryotic genome assemblies.

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July 7, 2019

Next-generation sequencing-based detection of germline L1-mediated transductions.

While active LINE-1 (L1) elements possess the ability to mobilize flanking sequences to different genomic loci through a process termed transduction influencing genomic content and structure, an approach for detecting polymorphic germline non-reference transductions in massively-parallel sequencing data has been lacking.Here we present the computational approach TIGER (Transduction Inference in GERmline genomes), enabling the discovery of non-reference L1-mediated transductions by combining L1 discovery with detection of unique insertion sequences and detailed characterization of insertion sites. We employed TIGER to characterize polymorphic transductions in fifteen genomes from non-human primate species (chimpanzee, orangutan and rhesus macaque), as well as in a human genome. We achieved high accuracy as confirmed by PCR and two single molecule DNA sequencing techniques, and uncovered differences in relative rates of transduction between primate species.By enabling detection of polymorphic transductions, TIGER makes this form of relevant structural variation amenable for population and personal genome analysis.

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