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Auto Tag: Comparative analysis

April 21, 2020  |  

Deep convolutional neural networks for accurate somatic mutation detection.

Accurate detection of somatic mutations is still a challenge in cancer analysis. Here we present NeuSomatic, the first convolutional neural network approach for somatic mutation detection, which significantly outperforms previous methods on different sequencing platforms, sequencing strategies, and tumor purities. NeuSomatic summarizes sequence alignments into small matrices and incorporates more than a hundred features to capture mutation signals effectively. It can be used universally as a stand-alone somatic mutation detection method or with an ensemble of existing methods to achieve the highest accuracy.

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April 21, 2020  |  

Sequence properties of certain GC rich avian genes, their origins and absence from genome assemblies: case studies.

More and more eukaryotic genomes are sequenced and assembled, most of them presented as a complete model in which missing chromosomal regions are filled by Ns and where a few chromosomes may be lacking. Avian genomes often contain sequences with high GC content, which has been hypothesized to be at the origin of many missing sequences in these genomes. We investigated features of these missing sequences to discover why some may not have been integrated into genomic libraries and/or sequenced.The sequences of five red jungle fowl cDNA models with high GC content were used as queries to search publicly available datasets of Illumina and Pacbio sequencing reads. These were used to reconstruct the leptin, TNFa, MRPL52, PCP2 and PET100 genes, all of which are absent from the red jungle fowl genome model. These gene sequences displayed elevated GC contents, had intron sizes that were sometimes larger than non-avian orthologues, and had non-coding regions that contained numerous tandem and inverted repeat sequences with motifs able to assemble into stable G-quadruplexes and intrastrand dyadic structures. Our results suggest that Illumina technology was unable to sequence the non-coding regions of these genes. On the other hand, PacBio technology was able to sequence these regions, but with dramatically lower efficiency than would typically be expected.High GC content was not the principal reason why numerous GC-rich regions of avian genomes are missing from genome assembly models. Instead, it is the presence of tandem repeats containing motifs capable of assembling into very stable secondary structures that is likely responsible.

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April 21, 2020  |  

The Impact of cDNA Normalization on Long-Read Sequencing of a Complex Transcriptome

Normalization of cDNA is widely used to improve the coverage of rare transcripts in analysis of transcriptomes employing next-generation sequencing. Recently, long-read technology has been emerging as a powerful tool for sequencing and construction of transcriptomes, especially for complex genomes containing highly similar transcripts and transcript-spliced isoforms. Here, we analyzed the transcriptome of sugarcane, with a highly polyploidy plant genome, by PacBio isoform sequencing (Iso-Seq) of two different cDNA library preparations, with and without a normalization step. The results demonstrated that, while the two libraries included many of the same transcripts, many longer transcripts were removed and many new generally shorter transcripts were detected by normalization. For the same input cDNA and the same data yield, the normalized library recovered more total transcript isoforms, number of predicted gene families and orthologous groups, resulting in a higher representation for the sugarcane transcriptome, compared to the non-normalized library. The non-normalized library, on the other hand, included a wider transcript length range with more longer transcripts above ~1.25 kb, more transcript isoforms per gene family and gene ontology terms per transcript. A large proportion of the unique transcripts comprising ~52% of the normalized library were expressed at a lower level than the unique transcripts from the non-normalized library, across three tissue types tested including leaf, stalk and root. About 83% of the total 5,348 predicted long noncoding transcripts was derived from the normalized library, of which ~80% was derived from the lowly expressed fraction. Functional annotation of the unique transcripts suggested that each library enriched different functional transcript fractions. This demonstrated the complementation of the two approaches in obtaining a complete transcriptome of a complex genome at the sequencing depth used in this study.

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April 21, 2020  |  

Complete genome sequence analysis of the thermoacidophilic verrucomicrobial methanotroph “Candidatus Methylacidiphilum kamchatkense” strain Kam1 and comparison with its closest relatives.

The candidate genus “Methylacidiphilum” comprises thermoacidophilic aerobic methane oxidizers belonging to the Verrucomicrobia phylum. These are the first described non-proteobacterial aerobic methane oxidizers. The genes pmoCAB, encoding the particulate methane monooxygenase do not originate from horizontal gene transfer from proteobacteria. Instead, the “Ca. Methylacidiphilum” and the sister genus “Ca. Methylacidimicrobium” represent a novel and hitherto understudied evolutionary lineage of aerobic methane oxidizers. Obtaining and comparing the full genome sequences is an important step towards understanding the evolution and physiology of this novel group of organisms.Here we present the closed genome of “Ca. Methylacidiphilum kamchatkense” strain Kam1 and a comparison with the genomes of its two closest relatives “Ca. Methylacidiphilum fumariolicum” strain SolV and “Ca. Methylacidiphilum infernorum” strain V4. The genome consists of a single 2,2 Mbp chromosome with 2119 predicted protein coding sequences. Genome analysis showed that the majority of the genes connected with metabolic traits described for one member of “Ca. Methylacidiphilum” is conserved between all three genomes. All three strains encode class I CRISPR-cas systems. The average nucleotide identity between “Ca. M. kamchatkense” strain Kam1 and strains SolV and V4 is =95% showing that they should be regarded as separate species. Whole genome comparison revealed a high degree of synteny between the genomes of strains Kam1 and SolV. In contrast, comparison of the genomes of strains Kam1 and V4 revealed a number of rearrangements. There are large differences in the numbers of transposable elements found in the genomes of the three strains with 12, 37 and 80 transposable elements in the genomes of strains Kam1, V4 and SolV respectively. Genomic rearrangements and the activity of transposable elements explain much of the genomic differences between strains. For example, a type 1h uptake hydrogenase is conserved between strains Kam1 and SolV but seems to have been lost from strain V4 due to genomic rearrangements.Comparing three closed genomes of “Ca. Methylacidiphilum” spp. has given new insights into the evolution of these organisms and revealed large differences in numbers of transposable elements between strains, the activity of these explains much of the genomic differences between strains.

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April 21, 2020  |  

Denitrifying Bacteria Active in Woodchip Bioreactors at Low-Temperature Conditions.

Woodchip bioreactor technology removes nitrate from agricultural subsurface drainage by using denitrifying microorganisms. Although woodchip bioreactors have demonstrated success in many field locations, low water temperature can significantly limit bioreactor efficiency and performance. To improve bioreactor performance, it is important to identify the microbes responsible for nitrate removal at low temperature conditions. Therefore, in this study, we identified and characterized denitrifiers active at low-temperature conditions by using culture-independent and -dependent approaches. By comparative 16S rRNA (gene) analysis and culture isolation technique, Pseudomonas spp., Polaromonas spp., and Cellulomonas spp. were identified as being important bacteria responsible for denitrification in woodchip bioreactor microcosms at relatively low temperature conditions (15°C). Genome analysis of Cellulomonas sp. strain WB94 confirmed the presence of nitrite reductase gene nirK. Transcription levels of this nirK were significantly higher in the denitrifying microcosms than in the non-denitrifying microcosms. Strain WB94 was also capable of degrading cellulose and other complex polysaccharides. Taken together, our results suggest that Cellulomonas sp. denitrifiers could degrade woodchips to provide carbon source and electron donors to themselves and other denitrifiers in woodchip bioreactors at low-temperature conditions. By inoculating these denitrifiers (i.e., bioaugmentation), it might be possible to increase the nitrate removal rate of woodchip bioreactors at low-temperature conditions.

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April 21, 2020  |  

Chromosome-Level Alpaca Reference Genome VicPac3.1 Improves Genomic Insight Into the Biology of New World Camelids.

The development of high-quality chromosomally assigned reference genomes constitutes a key feature for understanding genome architecture of a species and is critical for the discovery of the genetic blueprints of traits of biological significance. South American camelids serve people in extreme environments and are important fiber and companion animals worldwide. Despite this, the alpaca reference genome lags far behind those available for other domestic species. Here we produced a chromosome-level improved reference assembly for the alpaca genome using the DNA of the same female Huacaya alpaca as in previous assemblies. We generated 190X Illumina short-read, 8X Pacific Biosciences long-read and 60X Dovetail Chicago® chromatin interaction scaffolding data for the assembly, used testis and skin RNAseq data for annotation, and cytogenetic map data for chromosomal assignments. The new assembly VicPac3.1 contains 90% of the alpaca genome in just 103 scaffolds and 76% of all scaffolds are mapped to the 36 pairs of the alpaca autosomes and the X chromosome. Preliminary annotation of the assembly predicted 22,462 coding genes and 29,337 isoforms. Comparative analysis of selected regions of the alpaca genome, such as the major histocompatibility complex (MHC), the region involved in the Minute Chromosome Syndrome (MCS) and candidate genes for high-altitude adaptations, reveal unique features of the alpaca genome. The alpaca reference genome VicPac3.1 presents a significant improvement in completeness, contiguity and accuracy over VicPac2 and is an important tool for the advancement of genomics research in all New World camelids.

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April 21, 2020  |  

A Phage-Like Plasmid Carrying blaKPC-2 Gene in Carbapenem-Resistant Pseudomonas aeruginosa.

Background: Lateral gene transfer plays a central role in the dissemination of carbapenem resistance in bacterial pathogens associated with nosocomial infections, mainly Enterobacteriaceae and Pseudomonas aeruginosa. Despite their clinical significance, there is little information regarding the mobile genetic elements and mechanism of acquisition and propagation of lateral genes in P. aeruginosa, and they remain largely unknown. Objectives: The present study characterized the genetic context of blaKPC-2 in carbapenem-resistant P. aeruginosa strain BH9. Methods:Pseudomonas aeruginosa BH9 sequencing was performed using the long-read PacBio SMRT platform and the Ion Proton System. De novo assembly was carried out using the SMRT pipeline and Canu, and gene prediction and annotation were performed using Prokka and RAST. Results:Pseudomonas aeruginosa BH9 exhibited a 7.1 Mb circular chromosome. However, the blaKPC-2 gene is located in an additional contig composed by a small plasmid pBH6 from P. aeruginosa strain BH6 and several phage-related genes. Further analysis revealed that the beginning and end of the contig contain identical sequences, supporting a circular plasmid structure. This structure spans 41,087 bp, exhibiting all the Mu-like phage landmarks. In addition, 5-bp direct repeats (GGATG) flanking the pBH6 ends were found, strongly indicating integration of the Mu-like phage into the pBH6 plasmid. Mu phages are commonly found in P. aeruginosa. However, for the first time showing a potential impact in shaping the vehicles of the dissemination of antimicrobial (e.g., plasmid pBH6) resistance genes in the Pseudomonas genus. Conclusion: pBH6 captured the Mu-like Phage BH9, creating a co-integrate pBH6::Phage BH9, and this phage-plasmid complex may represent novel case of a phage-like plasmid.

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April 21, 2020  |  

Prediction of Host-Specific Genes by Pan-Genome Analyses of the Korean Ralstonia solanacearum Species Complex.

The soil-borne pathogenic Ralstonia solanacearum species complex (RSSC) is a group of plant pathogens that is economically destructive worldwide and has a broad host range, including various solanaceae plants, banana, ginger, sesame, and clove. Previously, Korean RSSC strains isolated from samples of potato bacterial wilt were grouped into four pathotypes based on virulence tests against potato, tomato, eggplant, and pepper. In this study, we sequenced the genomes of 25 Korean RSSC strains selected based on these pathotypes. The newly sequenced genomes were analyzed to determine the phylogenetic relationships between the strains with average nucleotide identity values, and structurally compared via multiple genome alignment using Mauve software. To identify candidate genes responsible for the host specificity of the pathotypes, functional genome comparisons were conducted by analyzing pan-genome orthologous group (POG) and type III secretion system effectors (T3es). POG analyses revealed that a total of 128 genes were shared only in tomato-non-pathogenic strains, 8 genes in tomato-pathogenic strains, 5 genes in eggplant-non-pathogenic strains, 7 genes in eggplant-pathogenic strains, 1 gene in pepper-non-pathogenic strains, and 34 genes in pepper-pathogenic strains. When we analyzed T3es, three host-specific effectors were predicted: RipS3 (SKWP3) and RipH3 (HLK3) were found only in tomato-pathogenic strains, and RipAC (PopC) were found only in eggplant-pathogenic strains. Overall, we identified host-specific genes and effectors that may be responsible for virulence functions in RSSC in silico. The expected characters of those genes suggest that the host range of RSSC is determined by the comprehensive actions of various virulence factors, including effectors, secretion systems, and metabolic enzymes.

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April 21, 2020  |  

Reconstruction of the full-length transcriptome atlas using PacBio Iso-Seq provides insight into the alternative splicing in Gossypium australe.

Gossypium australe F. Mueller (2n?=?2x?=?26, G2 genome) possesses valuable characteristics. For example, the delayed gland morphogenesis trait causes cottonseed protein and oil to be edible while retaining resistance to biotic stress. However, the lack of gene sequences and their alternative splicing (AS) in G. australe remain unclear, hindering to explore species-specific biological morphogenesis.Here, we report the first sequencing of the full-length transcriptome of the Australian wild cotton species, G. australe, using Pacific Biosciences single-molecule long-read isoform sequencing (Iso-Seq) from the pooled cDNA of ten tissues to identify transcript loci and splice isoforms. We reconstructed the G. australe full-length transcriptome and identified 25,246 genes, 86 pre-miRNAs and 1468 lncRNAs. Most genes (12,832, 50.83%) exhibited two or more isoforms, suggesting a high degree of transcriptome complexity in G. australe. A total of 31,448 AS events in five major types were found among the 9944 gene loci. Among these five major types, intron retention was the most frequent, accounting for 68.85% of AS events. 29,718 polyadenylation sites were detected from 14,536 genes, 7900 of which have alternative polyadenylation sites (APA). In addition, based on our AS events annotations, RNA-Seq short reads from germinating seeds showed that differential expression of these events occurred during seed germination. Ten AS events that were randomly selected were further confirmed by RT-PCR amplification in leaf and germinating seeds.The reconstructed gene sequences and their AS in G. australe would provide information for exploring beneficial characteristics in G. australe.

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April 21, 2020  |  

Improved annotation of the domestic pig genome through integration of Iso-Seq and RNA-seq data.

Our understanding of the pig transcriptome is limited. RNA transcript diversity among nine tissues was assessed using poly(A) selected single-molecule long-read isoform sequencing (Iso-seq) and Illumina RNA sequencing (RNA-seq) from a single White cross-bred pig. Across tissues, a total of 67,746 unique transcripts were observed, including 60.5% predicted protein-coding, 36.2% long non-coding RNA and 3.3% nonsense-mediated decay transcripts. On average, 90% of the splice junctions were supported by RNA-seq within tissue. A large proportion (80%) represented novel transcripts, mostly produced by known protein-coding genes (70%), while 17% corresponded to novel genes. On average, four transcripts per known gene (tpg) were identified; an increase over current EBI (1.9 tpg) and NCBI (2.9 tpg) annotations and closer to the number reported in human genome (4.2 tpg). Our new pig genome annotation extended more than 6000 known gene borders (5′ end extension, 3′ end extension, or both) compared to EBI or NCBI annotations. We validated a large proportion of these extensions by independent pig poly(A) selected 3′-RNA-seq data, or human FANTOM5 Cap Analysis of Gene Expression data. Further, we detected 10,465 novel genes (81% non-coding) not reported in current pig genome annotations. More than 80% of these novel genes had transcripts detected in >?1 tissue. In addition, more than 80% of novel intergenic genes with at least one transcript detected in liver tissue had H3K4me3 or H3K36me3 peaks mapping to their promoter and gene body, respectively, in independent liver chromatin immunoprecipitation data. These validated results show significant improvement over current pig genome annotations.

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April 21, 2020  |  

Comparative analysis of the chicken IFITM locus by targeted genome sequencing reveals evolution of the locus and positive selection in IFITM1 and IFITM3.

The interferon-induced transmembrane (IFITM) protein family comprises a class of restriction factors widely characterised in humans for their potent antiviral activity. Their biological activity is well documented in several animal species, but their genetic variation and biological mechanism is less well understood, particularly in avian species.Here we report the complete sequence of the domestic chicken Gallus gallus IFITM locus from a wide variety of chicken breeds to examine the detailed pattern of genetic variation of the locus on chromosome 5, including the flanking genes ATHL1 and B4GALNT4. We have generated chIFITM sequences from commercial breeds (supermarket-derived chicken breasts), indigenous chickens from Nigeria (Nsukka) and Ethiopia, European breeds and inbred chicken lines from the Pirbright Institute, totalling of 206 chickens. Through mapping of genetic variants to the latest chIFITM consensus sequence our data reveal that the chIFITM locus does not show structural variation in the locus across the populations analysed, despite spanning diverse breeds from different geographic locations. However, single nucleotide variants (SNVs) in functionally important regions of the proteins within certain groups of chickens were detected, in particular the European breeds and indigenous birds from Ethiopia and Nigeria. In addition, we also found that two out of four SNVs located in the chIFITM1 (Ser36 and Arg77) and chIFITM3 (Val103) proteins were simultaneously under positive selection.Together these data suggest that IFITM genetic variation may contribute to the capacities of different chicken populations to resist virus infection.

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April 21, 2020  |  

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

Rapid innovation in sequencing technologies and improvement in assembly algorithms have enabled the creation of highly contiguous mammalian genomes. Here we report a chromosome-level assembly of the water buffalo (Bubalus bubalis) genome using single-molecule sequencing and chromatin conformation capture data. PacBio Sequel reads, with a mean length of 11.5?kb, helped to resolve repetitive elements and generate sequence contiguity. All five B. bubalis sub-metacentric chromosomes were correctly scaffolded with centromeres spanned. Although the index animal was partly inbred, 58% of the genome was haplotype-phased by FALCON-Unzip. This new reference genome improves the contig N50 of the previous short-read based buffalo assembly more than a thousand-fold and contains only 383 gaps. It surpasses the human and goat references in sequence contiguity and facilitates the annotation of hard to assemble gene clusters such as the major histocompatibility complex (MHC).

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April 21, 2020  |  

Comparative Phylogenomics, a Stepping Stone for Bird Biodiversity Studies

Birds are a group with immense availability of genomic resources, and hundreds of forthcoming genomes at the doorstep. We review recent developments in whole genome sequencing, phylogenomics, and comparative genomics of birds. Short read based genome assemblies are common, largely due to efforts of the Bird 10K genome project (B10K). Chromosome-level assemblies are expected to increase due to improved long-read sequencing. The available genomic data has enabled the reconstruction of the bird tree of life with increasing confidence and resolution, but challenges remain in the early splits of Neoaves due to their explosive diversification after the Cretaceous-Paleogene (K-Pg) event. Continued genomic sampling of the bird tree of life will not just better reflect their evolutionary history but also shine new light onto the organization of phylogenetic signal and conflict across the genome. The comparatively simple architecture of avian genomes makes them a powerful system to study the molecular foundation of bird specific traits. Birds are on the verge of becoming an extremely resourceful system to study biodiversity from the nucleotide up.

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April 21, 2020  |  

Adaptive Strategies in a Poly-Extreme Environment: Differentiation of Vegetative Cells in Serratia ureilytica and Resistance to Extreme Conditions.

Poly-extreme terrestrial habitats are often used as analogs to extra-terrestrial environments. Understanding the adaptive strategies allowing bacteria to thrive and survive under these conditions could help in our quest for extra-terrestrial planets suitable for life and understanding how life evolved in the harsh early earth conditions. A prime example of such a survival strategy is the modification of vegetative cells into resistant resting structures. These differentiated cells are often observed in response to harsh environmental conditions. The environmental strain (strain Lr5/4) belonging to Serratia ureilytica was isolated from a geothermal spring in Lirima, Atacama Desert, Chile. The Atacama Desert is the driest habitat on Earth and furthermore, due to its high altitude, it is exposed to an increased amount of UV radiation. The geothermal spring from which the strain was isolated is oligotrophic and the temperature of 54°C exceeds mesophilic conditions (15 to 45°C). Although the vegetative cells were tolerant to various environmental insults (desiccation, extreme pH, glycerol), a modified cell type was formed in response to nutrient deprivation, UV radiation and thermal shock. Scanning (SEM) and Transmission Electron Microscopy (TEM) analyses of vegetative cells and the modified cell structures were performed. In SEM, a change toward a circular shape with reduced size was observed. These circular cells possessed what appears as extra coating layers under TEM. The resistance of the modified cells was also investigated, they were resistant to wet heat, UV radiation and desiccation, while vegetative cells did not withstand any of those conditions. A phylogenomic analysis was undertaken to investigate the presence of known genes involved in dormancy in other bacterial clades. Genes related to spore-formation in Myxococcus and Firmicutes were found in S. ureilytica Lr5/4 genome; however, these genes were not enough for a full sporulation pathway that resembles either group. Although, the molecular pathway of cell differentiation in S. ureilytica Lr5/4 is not fully defined, the identified genes may contribute to the modified phenotype in the Serratia genus. Here, we show that a modified cell structure can occur as a response to extremity in a species that was previously not known to deploy this strategy. This strategy may be widely spread in bacteria, but only expressed under poly-extreme environmental conditions.

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April 21, 2020  |  

Characterization of an NDM-5 carbapenemase-producing Escherichia coli ST156 isolate from a poultry farm in Zhejiang, China.

The emergence of carbapenem-resistant Enterobacteriaceae strains has posed a severe threat to public health in recent years. The mobile elements carrying the New Delhi metallo-ß-lactqtamase (NDM) gene have been regarded as the major mechanism leading to the rapid increase of carbapenem-resistant Enterobacteriaceae strains isolated from clinics and animals.We describe an NDM-5-producing Escherichia coli strain, ECCRA-119 (sequence type 156 [ST156]), isolated from a poultry farm in Zhejiang, China. ECCRA-119 is a multidrug-resistant (MDR) isolate that exhibited resistance to 27 antimicrobial compounds, including imipenem and meropenem, as detected by antimicrobial susceptibility testing (AST). The complete genome sequence of the ECCRA-119 isolate was also obtained using the PacBio RS II platform. Eleven acquired resistance genes were identified in the chromosome; four were detected in plasmid pTB201, while six were detected in plasmid pTB202. Importantly, the carbapenem-resistant gene blaNDM-5 was detected in the IncX3 plasmid pTB203. In addition, seven virulence genes and one metal-resistance gene were also detected. The results of conjugation experiments and the transfer regions identification indicated that the blaNDM-5-harboring plasmid pTB203 could be transferred between E. coli strains.The results reflected the severe bacterial resistance in a poultry farm in Zhejiang province and increased our understanding of the presence and transmission of the blaNDM-5 gene.

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