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Asset Tag: Sequel System

April 21, 2020

The history, genome and biology of NCTC 30: a non-pandemic Vibrio cholerae isolate from World War One.

The sixth global cholera pandemic lasted from 1899 to 1923. However, despite widespread fear of the disease and of its negative effects on troop morale, very few soldiers in the British Expeditionary Forces contracted cholera between 1914 and 1918. Here, we have revived and sequenced the genome of NCTC 30, a 102-year-old Vibrio cholerae isolate, which we believe is the oldest publicly available live V. cholerae strain in existence. NCTC 30 was isolated in 1916 from a British soldier convalescent in Egypt. We found that this strain does not encode cholera toxin, thought to be necessary to cause cholera, and is not part of V. cholerae lineages responsible for the pandemic disease. We also show that NCTC 30, which predates the introduction of penicillin-based antibiotics, harbours a functional ß-lactamase antibiotic resistance gene. Our data corroborate and provide molecular explanations for previous phenotypic studies of NCTC 30 and provide a new high-quality genome sequence for historical, non-pandemic V. cholerae.

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April 21, 2020

Competition between mobile genetic elements drives optimization of a phage-encoded CRISPR-Cas system: insights from a natural arms race.

CRISPR-Cas systems function as adaptive immune systems by acquiring nucleotide sequences called spacers that mediate sequence-specific defence against competitors. Uniquely, the phage ICP1 encodes a Type I-F CRISPR-Cas system that is deployed to target and overcome PLE, a mobile genetic element with anti-phage activity in Vibrio cholerae. Here, we exploit the arms race between ICP1 and PLE to examine spacer acquisition and interference under laboratory conditions to reconcile findings from wild populations. Natural ICP1 isolates encode multiple spacers directed against PLE, but we find that single spacers do not interfere equally with PLE mobilization. High-throughput sequencing to assay spacer acquisition reveals that ICP1 can also acquire spacers that target the V. cholerae chromosome. We find that targeting the V. cholerae chromosome proximal to PLE is sufficient to block PLE and is dependent on Cas2-3 helicase activity. We propose a model in which indirect chromosomal spacers are able to circumvent PLE by Cas2-3-mediated processive degradation of the V. cholerae chromosome before PLE mobilization. Generally, laboratory-acquired spacers are much more diverse than the subset of spacers maintained by ICP1 in nature, showing how evolutionary pressures can constrain CRISPR-Cas targeting in ways that are often not appreciated through in vitro analyses. This article is part of a discussion meeting issue ‘The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems’.

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April 21, 2020

The sequence and de novo assembly of Oxygymnocypris stewartii genome.

Animal genomes in the Qinghai-Tibetan Plateau provide valuable resources for scientists to understand the molecular mechanism of environmental adaptation. Tibetan fish species play essential roles in the local ecology; however, the genomic information for native fishes was still insufficient. Oxygymnocypris stewartii, belonging to Oxygymnocypris genus, Schizothoracinae subfamily, is a native fish in the Tibetan plateau living within the elevation from roughly 3,000?m to 4,200?m. In this report, PacBio and Illumina sequencing platform were used to generate ~385.3?Gb genomic sequencing data. A genome of about 1,849.2?Mb was obtained with a contig N50 length of 257.1?kb. More than 44.5% of the genome were identified as repetitive elements, and 46,400 protein-coding genes were annotated in the genome. The assembled genome can be used as a reference for future population genetic studies of O. stewartii and will improve our understanding of high altitude adaptation of fishes in the Qinghai-Tibetan Plateau.

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April 21, 2020

Identification of putative genes for polyphenol biosynthesis in olive fruits and leaves using full-length transcriptome sequencing.

Olive (Olea europaea) is a rich source of valuable bioactive polyphenols, which has attracted widespread interest. In this study, we combined targeted metabolome, Pacbio ISOseq transcriptome, and Illumina RNA-seq transcriptome to investigate the association between polyphenols and gene expression in the developing olive fruits and leaves. A total of 12 main polyphenols were measured, and 122 transcripts of 17 gene families, 101 transcripts of 9 gene families, and 106 transcripts of 6 gene families that encode for enzymes involved in flavonoid, oleuropein, and hydroxytyrosol biosynthesis were separately identified. Additionally, 232 alternative splicing events of 18 genes related to polyphenol synthesis were analyzed. This is the first time that the third generations of full-length transcriptome technology were used to study the gene expression pattern of olive fruits and leaves. The results of transcriptome combined with targeted metabolome can help us better understand the polyphenol biosynthesis pathways in the olive.Copyright © 2019 Elsevier Ltd. All rights reserved.

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April 21, 2020

Distribution and antimicrobial activity of lactic acid bacteria from raw camel milk.

Consumer demand for natural pathogen-control agents for substitution of synthetic food preservatives and traditional antibiotics is increasing. This study aimed to reveal the distribution of lactic acid bacteria (LAB) in raw camel milk and to characterize their antimicrobial traits. The genetic identification by 16S rRNA sequencing of 58 LAB isolates showed the predominance of Enterococcus (24.2%), Lactococcus (22.4%) and Pediococcus (20.7%) genera in raw camel milk. These genera exhibited inhibitory activity against a broad spectrum of Gram-positive and Gram-negative bacteria including multidrug-resistant Salmonella. Among these LAB, two isolates-identified as Pediococcus pentosaceus CM16 and Lactobacillus brevis CM22-were selected for their strong bacteriocinogenic anti-listerial activity estimated at 1600 and 800 AU/mL, respectively. The bacteriocins produced were partially purified by ammonium sulphate precipitation and gel filtration and then biochemically characterized. The proteinaceous nature of bacteriocins was confirmed by the susceptibility to enzymes. These bacteriocins showed significant technological characteristics such as heat-resistance, and stability over a wide range of pH (2.0-10.0). In conclusion, these results indicated that Pediococcus pentosaceus CM16 and Lactobacillus brevis CM22 could be useful as potential probiotics. Moreover, their partially purified bacteriocins may play an important role as food preservatives and feed additives. To our knowledge, this is the first report describing the distribution of LAB population in raw camel milk and the characterization of their bacteriocins from the Arabian Peninsula of western Asia.

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April 21, 2020

The Single-molecule long-read sequencing of Scylla paramamosain.

Scylla paramamosain is an important aquaculture crab, which has great economical and nutritional value. To the best of our knowledge, few full-length crab transcriptomes are available. In this study, a library composed of 12 different tissues including gill, hepatopancreas, muscle, cerebral ganglion, eyestalk, thoracic ganglia, intestine, heart, testis, ovary, sperm reservoir, and hemocyte was constructed and sequenced using Pacific Biosciences single-molecule real-time (SMRT) long-read sequencing technology. A total of 284803 full-length non-chimeric reads were obtained, from which 79005 high-quality unique transcripts were obtained after error correction and sequence clustering and redundant. Additionally, a total of 52544 transcripts were annotated against protein database (NCBI nonredundant, Swiss-Prot, KOG, and KEGG database). A total of 23644 long non-coding RNAs (lncRNAs) and 131561 simple sequence repeats (SSRs) were identified. Meanwhile, the isoforms of many genes were also identified in this study. Our study provides a rich set of full-length cDNA sequences for S. paramamosain, which will greatly facilitate S. paramamosain research.

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April 21, 2020

A high-quality genome of Eragrostis curvula grass provides insights into Poaceae evolution and supports new strategies to enhance forage quality.

The Poaceae constitute a taxon of flowering plants (grasses) that cover almost all Earth’s inhabitable range and comprises some of the genera most commonly used for human and animal nutrition. Many of these crops have been sequenced, like rice, Brachypodium, maize and, more recently, wheat. Some important members are still considered orphan crops, lacking a sequenced genome, but having important traits that make them attractive for sequencing. Among these traits is apomixis, clonal reproduction by seeds, present in some members of the Poaceae like Eragrostis curvula. A de novo, high-quality genome assembly and annotation for E. curvula have been obtained by sequencing 602?Mb of a diploid genotype using a strategy that combined long-read length sequencing with chromosome conformation capture. The scaffold N50 for this assembly was 43.41?Mb and the annotation yielded 56,469 genes. The availability of this genome assembly has allowed us to identify regions associated with forage quality and to develop strategies to sequence and assemble the complex tetraploid genotypes which harbor the apomixis control region(s). Understanding and subsequently manipulating the genetic drivers underlying apomixis could revolutionize agriculture.

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April 21, 2020

Mitogenome types of two Lentinula edodes sensu lato populations in China.

China has two populations of Lentinula edodes sensu lato as follows: L. edodes sensu stricto and an unexcavated morphological species respectively designated as A and B. In a previous study, we found that the nuclear types of the two populations are distinct and that both have two branches (A1, A2, B1 and B2) based on the internal transcribed spacer 2 (ITS2) sequence. In this paper, their mitogenome types were studied by resequencing 20 of the strains. The results show that the mitogenome type (mt) of ITS2-A1 was mt-A1, that of ITS2-A2 was mt-A2, and those of ITS2-B1 and ITS2-B2 were mt-B. The strains with heterozygous ITS2 types had one mitogenome type, and some strains possessed a recombinant mitogenome. This indicated that there may be frequent genetic exchanges between the two populations and both nuclear and mitochondrial markers were necessary to identify the strains of L. edodes sensu lato. In addition, by screening SNP diversity and comparing four complete mitogenomes among mt-A1, mt-A2 and mt-B, the cob, cox3, nad2, nad3, nad4, nad5, rps3 and rrnS genes could be used to identify mt-A and mt-B and that the cox1, nad1 and rrnL genes could be used to identify mt-A1, mt-A2 and mt-B.

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April 21, 2020

Long-read assembly of the Chinese rhesus macaque genome and identification of ape-specific structural variants.

We present a high-quality de novo genome assembly (rheMacS) of the Chinese rhesus macaque (Macaca mulatta) using long-read sequencing and multiplatform scaffolding approaches. Compared to the current Indian rhesus macaque reference genome (rheMac8), rheMacS increases sequence contiguity 75-fold, closing 21,940 of the remaining assembly gaps (60.8 Mbp). We improve gene annotation by generating more than two million full-length transcripts from ten different tissues by long-read RNA sequencing. We sequence resolve 53,916 structural variants (96% novel) and identify 17,000 ape-specific structural variants (ASSVs) based on comparison to ape genomes. Many ASSVs map within ChIP-seq predicted enhancer regions where apes and macaque show diverged enhancer activity and gene expression. We further characterize a subset that may contribute to ape- or great-ape-specific phenotypic traits, including taillessness, brain volume expansion, improved manual dexterity, and large body size. The rheMacS genome assembly serves as an ideal reference for future biomedical and evolutionary studies.

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April 21, 2020

Metatranscriptomic evidence for classical and RuBisCO-mediated CO2 reduction to methane facilitated by direct interspecies electron transfer in a methanogenic system.

In a staged anaerobic fluidized-bed ceramic membrane bioreactor, metagenomic and metatranscriptomic analyses were performed to decipher the microbial interactions on the granular activated carbon. Metagenome bins, representing the predominating microbes in the bioreactor: syntrophic propionate-oxidizing bacteria (SPOB), acetoclastic Methanothrix concilii, and exoelectrogenic Geobacter lovleyi, were successfully recovered for the reconstruction and analysis of metabolic pathways involved in the transformation of fatty acids to methane. In particular, SPOB degraded propionate into acetate, which was further converted into methane and CO2 by M. concilii via the acetoclastic methanogenesis. Concurrently, G. lovleyi oxidized acetate into CO2, releasing electrons into the extracellular environment. By accepting these electrons through direct interspecies electron transfer (DIET), M. concilii was capable of performing CO2 reduction for further methane formation. Most notably, an alternative RuBisCO-mediated CO2 reduction (the reductive hexulose-phosphate (RHP) pathway) is transcriptionally-active in M. concilii. This RHP pathway enables M. concilii dominance and energy gain by carbon fixation and methanogenesis, respectively via a methyl-H4MPT intermediate, constituting the third methanogenesis route. The complete acetate reduction (2 mole methane formation/1 mole acetate consumption), coupling of acetoclastic methanogenesis and two CO2 reduction pathways, are thermodynamically favorable even under very low substrate condition (down to to 10-5?M level). Such tight interactions via both mediated and direct interspecies electron transfer (MIET and DIET), induced by the conductive GAC promote the overall efficiency of bioenergy processes.

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April 21, 2020

Uncovering the biosynthetic potential of rare metagenomic DNA using co-occurrence network analysis of targeted sequences.

Sequencing of DNA extracted from environmental samples can provide key insights into the biosynthetic potential of uncultured bacteria. However, the high complexity of soil metagenomes, which can contain thousands of bacterial species per gram of soil, imposes significant challenges to explore secondary metabolites potentially produced by rare members of the soil microbiome. Here, we develop a targeted sequencing workflow termed CONKAT-seq (co-occurrence network analysis of targeted sequences) that detects physically clustered biosynthetic domains, a hallmark of bacterial secondary metabolism. Following targeted amplification of conserved biosynthetic domains in a highly partitioned metagenomic library, CONKAT-seq evaluates amplicon co-occurrence patterns across library subpools to identify chromosomally clustered domains. We show that a single soil sample can contain more than a thousand uncharacterized biosynthetic gene clusters, most of which originate from low frequency genomes which are practically inaccessible through untargeted sequencing. CONKAT-seq allows scalable exploration of largely untapped biosynthetic diversity across multiple soils, and can guide the discovery of novel secondary metabolites from rare members of the soil microbiome.

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April 21, 2020

CRISPR/CAS9 targeted CAPTURE of mammalian genomic regions for characterization by NGS.

The robust detection of structural variants in mammalian genomes remains a challenge. It is particularly difficult in the case of genetically unstable Chinese hamster ovary (CHO) cell lines with only draft genome assemblies available. We explore the potential of the CRISPR/Cas9 system for the targeted capture of genomic loci containing integrated vectors in CHO-K1-based cell lines followed by next generation sequencing (NGS), and compare it to popular target-enrichment sequencing methods and to whole genome sequencing (WGS). Three different CRISPR/Cas9-based techniques were evaluated; all of them allow for amplification-free enrichment of target genomic regions in the range from 5 to 60 fold, and for recovery of ~15 kb-long sequences with no sequencing artifacts introduced. The utility of these protocols has been proven by the identification of transgene integration sites and flanking sequences in three CHO cell lines. The long enriched fragments helped to identify Escherichia coli genome sequences co-integrated with vectors, and were further characterized by Whole Genome Sequencing (WGS). Other advantages of CRISPR/Cas9-based methods are the ease of bioinformatics analysis, potential for multiplexing, and the production of long target templates for real-time sequencing.

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April 21, 2020

Genome-wide systematic identification of methyltransferase recognition and modification patterns.

Genome-wide analysis of DNA methylation patterns using single molecule real-time DNA sequencing has boosted the number of publicly available methylomes. However, there is a lack of tools coupling methylation patterns and the corresponding methyltransferase genes. Here we demonstrate a high-throughput method for coupling methyltransferases with their respective motifs, using automated cloning and analysing the methyltransferases in vectors carrying a strain-specific cassette containing all potential target sites. To validate the method, we analyse the genomes of the thermophile Moorella thermoacetica and the mesophile Acetobacterium woodii, two acetogenic bacteria having substantially modified genomes with 12 methylation motifs and a total of 23 methyltransferase genes. Using our method, we characterize the 23 methyltransferases, assign motifs to the respective enzymes and verify activity for 11 of the 12 motifs.

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April 21, 2020

Modulation of metabolome and bacterial community in whole crop corn silage by inoculating homofermentative Lactobacillus plantarum and heterofermentative Lactobacillus buchneri.

The present study investigated the species level based microbial community and metabolome in corn silage inoculated with or without homofermentative Lactobacillus plantarum and heterofermentative Lactobacillus buchneri using the PacBio SMRT Sequencing and time-of-flight mass spectrometry (GC-TOF/MS). Chopped whole crop corn was treated with (1) deionized water (control), (2) Lactobacillus plantarum, or (3) Lactobacillus buchneri. The chopped whole crop corn was ensiled in vacuum-sealed polyethylene bags containing 300 g of fresh forge for 90 days, with three replicates for each treatment. The results showed that a total of 979 substances were detected, and 316 different metabolites were identified. Some metabolites with antimicrobial activity were detected in whole crop corn silage, such as catechol, 3-phenyllactic acid, 4-hydroxybenzoic acid, azelaic acid, 3,4-dihydroxybenzoic acid and 4-hydroxycinnamic acid. Catechol, pyrogallol and ferulic acid with antioxidant property, 4-hydroxybutyrate with nervine activity, and linoleic acid with cholesterol lowering effects, were detected in present study. In addition, a flavoring agent of myristic acid and a depression mitigation substance of phenylethylamine were also found in this study. Samples treated with inoculants presented more biofunctional metabolites of organic acids, amino acids and phenolic acids than untreated samples. The Lactobacillus species covered over 98% after ensiling, and were mainly comprised by the L. acetotolerans, L. silagei, L. parafarraginis, L. buchneri and L. odoratitofui. As compared to the control silage, inoculation of L. plantarum increased the relative abundances of L. acetotolerans, L. buchneri and L. parafarraginis, and a considerable decline in the proportion of L. silagei was observed; whereas an obvious decrease in L. acetotolerans and increases in L. odoratitofui and L. farciminis were observed in the L. buchneri inoculated silage. Therefore, inoculation of L. plantarum and L. buchneri regulated the microbial composition and metabolome of the corn silage with different behaviors. The present results indicated that profiling of silage microbiome and metabolome might improve our current understanding of the biological process underlying silage formation.

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April 21, 2020

Extensive intraspecific gene order and gene structural variations in upland cotton cultivars.

Multiple cotton genomes (diploid and tetraploid) have been assembled. However, genomic variations between cultivars of allotetraploid upland cotton (Gossypium hirsutum L.), the most widely planted cotton species in the world, remain unexplored. Here, we use single-molecule long read and Hi-C sequencing technologies to assemble genomes of the two upland cotton cultivars TM-1 and zhongmiansuo24 (ZM24). Comparisons among TM-1 and ZM24 assemblies and the genomes of the diploid ancestors reveal a large amount of genetic variations. Among them, the top three longest structural variations are located on chromosome A08 of the tetraploid upland cotton, which account for ~30% total length of this chromosome. Haplotype analyses of the mapping population derived from these two cultivars and the germplasm panel show suppressed recombination rates in this region. This study provides additional genomic resources for the community, and the identified genetic variations, especially the reduced meiotic recombination on chromosome A08, will help future breeding.

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