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Asset Tag: Next gen sequencing

April 21, 2020

Decreased metabolism and increased tolerance to extreme environments in Staphylococcus warneri during long-term spaceflight.

Many studies have shown that the space environment can affect bacteria by causing a range of mutations. However, to date, few studies have explored the effects of long-term spaceflight (>1 month) on bacteria. In this study, a Staphylococcus warneri strain that was isolated from the Shenzhou-10 spacecraft and had experienced a spaceflight (15 days) was carried into space again. After a 64-day flight, combined phenotypic, genomic, transcriptomic, and proteomic analyses were performed to compare the influence of the two spaceflights on this bacterium. Compared with short-term spaceflight, long-term spaceflight increased the biofilm formation ability of S. warneri and the cell wall resistance to external environmental stress but reduced the sensitivity to chemical stimulation. Further analysis showed that these changes might be associated with the significantly upregulated gene expression of the phosphotransferase system, which regulates the metabolism of sugars, including glucose, mannose, fructose, and cellobiose. The mutation of S. warneri caused by the 15-day spaceflight was limited at the phenotype and gene level after cultivation on the ground. After 79 days of spaceflight, significant changes in S. warneri were observed. The phosphotransferase system of S. warneri was upregulated by long-term space stimulation, which resulted in a series of changes in the cell wall, biofilm, and chemical sensitivity, thus enhancing the resistance and adaptability of the bacterium to the external environment. © 2019 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

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April 21, 2020

Profiling the genome-wide landscape of tandem repeat expansions.

Tandem repeat (TR) expansions have been implicated in dozens of genetic diseases, including Huntington’s Disease, Fragile X Syndrome, and hereditary ataxias. Furthermore, TRs have recently been implicated in a range of complex traits, including gene expression and cancer risk. While the human genome harbors hundreds of thousands of TRs, analysis of TR expansions has been mainly limited to known pathogenic loci. A major challenge is that expanded repeats are beyond the read length of most next-generation sequencing (NGS) datasets and are not profiled by existing genome-wide tools. We present GangSTR, a novel algorithm for genome-wide genotyping of both short and expanded TRs. GangSTR extracts information from paired-end reads into a unified model to estimate maximum likelihood TR lengths. We validate GangSTR on real and simulated data and show that GangSTR outperforms alternative methods in both accuracy and speed. We apply GangSTR to a deeply sequenced trio to profile the landscape of TR expansions in a healthy family and validate novel expansions using orthogonal technologies. Our analysis reveals that healthy individuals harbor dozens of long TR alleles not captured by current genome-wide methods. GangSTR will likely enable discovery of novel disease-associated variants not currently accessible from NGS. © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.

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April 21, 2020

A new full-length circular DNA sequencing method for viral-sized genomes reveals that RNAi transgenic plants provoke a shift in geminivirus populations in the field.

We present a new method, CIDER-Seq (Circular DNA Enrichment sequencing) for the unbiased enrichment and long-read sequencing of viral-sized circular DNA molecules. We used CIDER-Seq to produce single-read full-length virus genomes for the first time. CIDER-Seq combines PCR-free virus enrichment with Single Molecule Real Time sequencing and a new sequence de-concatenation algorithm. We apply our technique to produce >1200 full-length, highly accurate geminivirus genomes from RNAi-transgenic and control plants in a field trial in Kenya. Using CIDER-Seq we can demonstrate for the first time that the expression of antiviral double-stranded RNA (dsRNA) in transgenic plants causes a consistent shift in virus populations towards species sharing low homology to the transgene derived dsRNA. Our method and its application in an economically important crop plant opens new possibilities in periodic virus sequence surveillance and accurate profiling of diverse circular DNA elements.

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April 21, 2020

Shared and unique microbes between Small hive beetles (Aethina tumida) and their honey bee hosts.

The small hive beetle (SHB) is an opportunistic parasite that feeds on bee larvae, honey, and pollen. While SHBs can also feed on fruit and other plant products, like its plant-feeding relatives, SHBs prefer to feed on hive resources and only reproduce inside bee colonies. As parasites, SHBs are inevitably exposed to bee-associated microbes, either directly from the bees or from the hive environment. These microbes have unknown impacts on beetles, nor is it known how extensively beetles transfer microbes among their bee hosts. To identify sets of beetle microbes and the transmission of microbes from bees to beetles, a metagenomic analysis was performed. We identified sets of herbivore-associated bacteria, as well as typical bee symbiotic bacteria for pollen digestion, in SHB larvae and adults. Deformed wing virus was highly abundant in beetles, which colonize SHBs as suggested by a controlled feeding trial. Our data suggest SHBs are vectors for pathogen transmission among bees and between colonies. The dispersal of host pathogens by social parasites via floral resources and the hive environment increases the threats of these parasites to honey bees. © 2019 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

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April 21, 2020

Full-length 16S rRNA gene classification of Atlantic salmon bacteria and effects of using different 16S variable regions on community structure analysis.

Understanding fish-microbial relationships may be of great value for fish producers as fish growth, development and welfare are influenced by the microbial community associated with the rearing systems and fish surfaces. Accurate methods to generate and analyze these microbial communities would be an important tool to help improve understanding of microbial effects in the industry. In this study, we performed taxonomic classification and determination of operational taxonomic units on Atlantic salmon microbiota by taking advantage of full-length 16S rRNA gene sequences. Skin mucus was dominated by the genera Flavobacterium and Psychrobacter. Intestinal samples were dominated by the genera Carnobacterium, Aeromonas, Mycoplasma and by sequences assigned to the order Clostridiales. Applying Sanger sequencing on the full-length bacterial 16S rRNA gene from the pool of 46 isolates obtained in this study showed a clear assignment of the PacBio full-length bacterial 16S rRNA gene sequences down to the genus level. One of the bottlenecks in comparing microbial profiles is that different studies use different 16S rRNA gene regions. Comparisons of sequence assignments between full-length and in silico derived variable 16S rRNA gene regions showed different microbial profiles with variable effects between phylogenetic groups and taxonomic ranks. © 2019 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

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April 21, 2020

Sensitivity to the two peptide bacteriocin plantaricin EF is dependent on CorC, a membrane-bound, magnesium/cobalt efflux protein.

Lactic acid bacteria produce a variety of antimicrobial peptides known as bacteriocins. Most bacteriocins are understood to kill sensitive bacteria through receptor-mediated disruptions. Here, we report on the identification of the Lactobacillus plantarum plantaricin EF (PlnEF) receptor. Spontaneous PlnEF-resistant mutants of the PlnEF-indicator strain L. plantarum NCIMB 700965 (LP965) were isolated and confirmed to maintain cellular ATP levels in the presence of PlnEF. Genome comparisons resulted in the identification of a single mutated gene annotated as the membrane-bound, magnesium/cobalt efflux protein CorC. All isolates contained a valine (V) at position 334 instead of a glycine (G) in a cysteine-ß-synthase domain at the C-terminal region of CorC. In silico template-based modeling of this domain indicated that the mutation resides in a loop between two ß-strands. The relationship between PlnEF, CorC, and metal homeostasis was supported by the finding that PlnEF-resistance was lost when PlnEF was applied together with high concentrations of Mg2+ , Co2+ , Zn2+ , or Cu2+ . Lastly, PlnEF sensitivity was increased upon heterologous expression of LP965 corC but not the G334V CorC mutant in the PlnEF-resistant strain Lactobacillus casei BL23. These results show that PlnEF kills sensitive bacteria by targeting CorC. © 2019 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

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April 21, 2020

Genome and transcriptome analysis of Bacillus velezensisBS-37, an efficient surfactin producer from glycerol, in response to d-/l-leucine.

Surfactin is one of the most widely studied biosurfactants due to its many potential applications in different fields. In the present study, Bacillus velezensis BS-37, initially identified as a strain of Bacillus subtilis, was used to efficiently produce surfactin with the addition of glycerol, an inexpensive by-product of biodiesel production. After 36 hr of growth in glycerol medium, the total surfactin concentration reached more than 1,000 mg/L, which was two times higher than that in sucrose medium. Moreover, the addition of l- and d-Leu to the culture medium had opposite effects on surfactin production by BS-37. While surfactin production increased significantly to nearly 2,000 mg/L with the addition of 10 mM l-Leu, it was dramatically reduced to about 250 mg/L with the addition of 10 mM d-Leu. To systemically elucidate the mechanisms influencing the efficiency of this biosynthesis process, we sequenced the genome of BS-37 and analyzed changes of the transcriptome in glycerol medium in response to d-/l-leucine. The RPKM analysis of the transcriptome of BS-37 showed that the transcription levels of genes encoding modular surfactin synthase, the glycerol utilization pathway, and branched-chain amino acid (BCAA) synthesis pathways were all at a relatively high level, which may offered an explanation why this strain can efficiently use glycerol to produce surfactin with a high yield. Neither l-Leu nor d-Leu had a significant effect on the expression of genes in these pathways, indicating that l-Leu plays an important role as a precursor or substrate involved in surfactin production, while d-Leu appears to act as a competitive inhibitor. The results of the present study provide new insights into the synthesis of surfactin and ways of its regulation, and enrich the genomic and transcriptomic resources available for the construction of high-producing strains. © 2019 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

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April 21, 2020

Aquella oligotrophica gen. nov. sp. nov.: A new member of the family Neisseriaceae isolated from laboratory tap water.

A bacterial strain designated as P08T was isolated from laboratory tap water during a water quality assessment in University of Malaya, Malaysia. The strain was a Gram-negative, rod-shaped, nonmotile, and aerobic bacterium. Complete genome of P08T comprised of a 2,820,660 bp chromosome with a G + C content of 36.43%. Both 16S rRNA phylogeny and phylogenetic tree inferred from the core gene matrix demonstrated that P08T formed a hitherto unknown subline within the family Neisseriaceae. Ortho average nucleotide identity (OrthoANI) values and the percentage of conserved proteins (POCP) calculated from complete genome sequence indicated low relatedness between P08T and its phylogenetic neighbors. Respiratory quinone analysis revealed Q-8 as the only detectable quinone. The predominant cellular fatty acids were identified as C14:0 , iso-C15:0 , and summed feature 3 (C16:1 ?7c/C16:1 ?6c). The polar lipids consisted of uncharacterized aminolipid, phosphatidylglycerol, and phosphatidylethanolamine. All aspects of phenotypic and phylogenetic data suggested that strain P08T represents a novel genus within family Neisseriaceae, for which the name Aquella gen. nov. is proposed. The type species of the genus is Aquella oligotrophica sp. nov., and the type strain is P08T (=LMG 29629T =DSM 100970T ). © 2019 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

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April 21, 2020

SMRT sequencing analysis reveals the full-length transcripts and alternative splicing patterns in Ananas comosus var. bracteatus.

Ananas comosus var. bracteatus is an herbaceous perennial monocot cultivated as an ornamental plant for its chimeric leaves. Because of its genomic complexity, and because no genomic information is available in the public GenBank database, the complete structure of the mRNA transcript is unclear and there are limited molecular mechanism studies for Ananas comosus var. bracteatus.Three size fractionated full-length cDNA libraries (1-2 kb, 2-3 kb, and 3-6 kb) were constructed and subsequently sequenced in five single-molecule real-time (SMRT) cells (2 cells, 2 cells, and 1 cell, respectively).In total, 19,838 transcripts were identified for alternative splicing (AS) analysis. Among them, 19,185 (96.7%) transcripts were functionally annotated. A total of 9,921 genes were identified by mapping the non-redundant isoforms to the reference genome. A total of 10,649 AS events were identified, the majority of which were intron retention events. The alternatively spliced genes had functions in the basic metabolism processes of the plant such as carbon metabolism, amino acid biosynthesis, and glycolysis. Fourteen genes related to chlorophyll biosynthesis were identified as having AS events. The distribution of the splicing sites and the percentage of conventional and non-canonical AS sites of the genes categorized in pathways related to the albino leaf phenotype (ko00860, ko00195, ko00196, and ko00710) varied greatly. The present results showed that there were 8,316 genes carrying at least one poly (A) site, which generated 21,873 poly (A) sites. These findings indicated that the quality of the gene structure and functional information of the obtained genome was greatly improved, which may facilitate further genetic study of Ananas comosus var. bracteatus.

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April 21, 2020

Biodiversity seen through the perspective of insects: 10 simple rules on methodological choices and experimental design for genomic studies.

Massively parallel DNA sequencing opens up opportunities for bridging multiple temporal and spatial dimensions in biodiversity research, thanks to its efficiency to recover millions of nucleotide polymorphisms. Here, we identify the current status, discuss the main challenges, and look into future perspectives on biodiversity genomics focusing on insects, which arguably constitute the most diverse and ecologically important group among all animals. We suggest 10 simple rules that provide a succinct step-by-step guide and best-practices to anyone interested in biodiversity research through the study of insect genomics. To this end, we review relevant literature on biodiversity and evolutionary research in the field of entomology. Our compilation is targeted at researchers and students who may not yet be specialists in entomology or molecular biology. We foresee that the genomic revolution and its application to the study of non-model insect lineages will represent a major leap to our understanding of insect diversity.

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April 21, 2020

Genes of the pig, Sus scrofa, reconstructed with EvidentialGene.

The pig is a well-studied model animal of biomedical and agricultural importance. Genes of this species, Sus scrofa, are known from experiments and predictions, and collected at the NCBI reference sequence database section. Gene reconstruction from transcribed gene evidence of RNA-seq now can accurately and completely reproduce the biological gene sets of animals and plants. Such a gene set for the pig is reported here, including human orthologs missing from current NCBI and Ensembl reference pig gene sets, additional alternate transcripts, and other improvements. Methodology for accurate and complete gene set reconstruction from RNA is used: the automated SRA2Genes pipeline of EvidentialGene project.

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April 21, 2020

Whole-genome comparisons of Penicillium spp. reveals secondary metabolic gene clusters and candidate genes associated with fungal aggressiveness during apple fruit decay.

Blue mold is a postharvest rot of pomaceous fruits caused by Penicillium expansum and a number of other Penicillium species. The genome of the highly aggressive P. expansum strain R19 was re-sequenced and analyzed together with the genome of the less aggressive P. solitum strain RS1. Whole genome scale similarities and differences were examined. A phylogenetic analysis of P. expansum, P. solitum, and several closely related Penicillium species revealed that the two pathogens isolated from decayed apple with blue mold symptoms are not each other’s closest relatives. Among a total of 10,560 and 10,672 protein coding sequences respectively, a comparative genomics analysis revealed 41 genes in P. expansum R19 and 43 genes in P. solitum RS1 that are unique to these two species. These genes may be associated with pome fruit-fungal interactions, subsequent decay processes, and mycotoxin accumulation. An intact patulin gene cluster consisting of 15 biosynthetic genes was identified in the patulin producing P. expansum strain R19, while only a remnant, seven-gene cluster was identified in the patulin-deficient P. solitum strain. However, P. solitum contained a large number of additional secondary metabolite gene clusters, indicating that this species has the potential capacity to produce an array of known as well as not-yet-identified products of possible toxicological or biotechnological interest.

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April 21, 2020

Hidden genomic evolution in a morphospecies-The landscape of rapidly evolving genes in Tetrahymena.

A morphospecies is defined as a taxonomic species based wholly on morphology, but often morphospecies consist of clusters of cryptic species that can be identified genetically or molecularly. The nature of the evolutionary novelty that accompanies speciation in a morphospecies is an intriguing question. Morphospecies are particularly common among ciliates, a group of unicellular eukaryotes that separates 2 kinds of nuclei-the silenced germline nucleus (micronucleus [MIC]) and the actively expressed somatic nucleus (macronucleus [MAC])-within a common cytoplasm. Because of their very similar morphologies, members of the Tetrahymena genus are considered a morphospecies. We explored the hidden genomic evolution within this genus by performing a comprehensive comparative analysis of the somatic genomes of 10 species and the germline genomes of 2 species of Tetrahymena. These species show high genetic divergence; phylogenomic analysis suggests that the genus originated about 300 million years ago (Mya). Seven universal protein domains are preferentially included among the species-specific (i.e., the youngest) Tetrahymena genes. In particular, leucine-rich repeat (LRR) genes make the largest contribution to the high level of genome divergence of the 10 species. LRR genes can be sorted into 3 different age groups. Parallel evolutionary trajectories have independently occurred among LRR genes in the different Tetrahymena species. Thousands of young LRR genes contain tandem arrays of exactly 90-bp exons. The introns separating these exons show a unique, extreme phase 2 bias, suggesting a clonal origin and successive expansions of 90-bp-exon LRR genes. Identifying LRR gene age groups allowed us to document a Tetrahymena intron length cycle. The youngest 90-bp exon LRR genes in T. thermophila are concentrated in pericentromeric and subtelomeric regions of the 5 micronuclear chromosomes, suggesting that these regions act as genome innovation centers. Copies of a Tetrahymena Long interspersed element (LINE)-like retrotransposon are very frequently found physically adjacent to 90-bp exon/intron repeat units of the youngest LRR genes. We propose that Tetrahymena species have used a massive exon-shuffling mechanism, involving unequal crossing over possibly in concert with retrotransposition, to create the unique 90-bp exon array LRR genes.

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April 21, 2020

Rapid antigen diversification through mitotic recombination in the human malaria parasite Plasmodium falciparum.

Malaria parasites possess the remarkable ability to maintain chronic infections that fail to elicit a protective immune response, characteristics that have stymied vaccine development and cause people living in endemic regions to remain at risk of malaria despite previous exposure to the disease. These traits stem from the tremendous antigenic diversity displayed by parasites circulating in the field. For Plasmodium falciparum, the most virulent of the human malaria parasites, this diversity is exemplified by the variant gene family called var, which encodes the major surface antigen displayed on infected red blood cells (RBCs). This gene family exhibits virtually limitless diversity when var gene repertoires from different parasite isolates are compared. Previous studies indicated that this remarkable genome plasticity results from extensive ectopic recombination between var genes during mitotic replication; however, the molecular mechanisms that direct this process to antigen-encoding loci while the rest of the genome remains relatively stable were not determined. Using targeted DNA double-strand breaks (DSBs) and long-read whole-genome sequencing, we show that a single break within an antigen-encoding region of the genome can result in a cascade of recombination events leading to the generation of multiple chimeric var genes, a process that can greatly accelerate the generation of diversity within this family. We also found that recombinations did not occur randomly, but rather high-probability, specific recombination products were observed repeatedly. These results provide a molecular basis for previously described structured rearrangements that drive diversification of this highly polymorphic gene family.

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April 21, 2020

Adding function to the genome of African Salmonella Typhimurium ST313 strain D23580.

Salmonella Typhimurium sequence type (ST) 313 causes invasive nontyphoidal Salmonella (iNTS) disease in sub-Saharan Africa, targeting susceptible HIV+, malarial, or malnourished individuals. An in-depth genomic comparison between the ST313 isolate D23580 and the well-characterized ST19 isolate 4/74 that causes gastroenteritis across the globe revealed extensive synteny. To understand how the 856 nucleotide variations generated phenotypic differences, we devised a large-scale experimental approach that involved the global gene expression analysis of strains D23580 and 4/74 grown in 16 infection-relevant growth conditions. Comparison of transcriptional patterns identified virulence and metabolic genes that were differentially expressed between D23580 versus 4/74, many of which were validated by proteomics. We also uncovered the S. Typhimurium D23580 and 4/74 genes that showed expression differences during infection of murine macrophages. Our comparative transcriptomic data are presented in a new enhanced version of the Salmonella expression compendium, SalComD23580: http://bioinf.gen.tcd.ie/cgi-bin/salcom_v2.pl. We discovered that the ablation of melibiose utilization was caused by three independent SNP mutations in D23580 that are shared across ST313 lineage 2, suggesting that the ability to catabolize this carbon source has been negatively selected during ST313 evolution. The data revealed a novel, to our knowledge, plasmid maintenance system involving a plasmid-encoded CysS cysteinyl-tRNA synthetase, highlighting the power of large-scale comparative multicondition analyses to pinpoint key phenotypic differences between bacterial pathovariants.

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