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April 21, 2020

A Newly Isolated Bacillus subtilis Strain Named WS-1 Inhibited Diarrhea and Death Caused by Pathogenic Escherichia coli in Newborn Piglets.

Bacillus subtilis is recognized as a safe and reliable human and animal probiotic and is associated with bioactivities such as production of vitamin and immune stimulation. Additionally, it has great potential to be used as an alternative to antimicrobial drugs, which is significant in the context of antibiotic abuse in food animal production. In this study, we isolated one strain of B. subtilis, named WS-1, from apparently healthy pigs growing with sick cohorts on one Escherichia coli endemic commercial pig farm in Guangdong, China. WS-1 can strongly inhibit the growth of pathogenic E. coli in vitro. The B. subtilis strain WS-1 showed typical Bacillus characteristics by endospore staining, biochemical test, enzyme activity analysis, and 16S rRNA sequence analysis. Genomic analysis showed that the B. subtilis strain WS-1 shares 100% genomic synteny with B. subtilis with a size of 4,088,167 bp. Importantly, inoculation of newborn piglets with 1.5 × 1010 CFU of B. subtilis strain WS-1 by oral feeding was able to clearly inhibit diarrhea (p < 0.05) and death (p < 0.05) caused by pathogenic E. coli in piglets. Furthermore, histopathological results showed that the WS-1 strain could protect small intestine from lesions caused by E. coli infection. Collectively, these findings suggest that the probiotic B. subtilis strain WS-1 acts as a potential biocontrol agent protecting pigs from pathogenic E. coli infection. Importance: In this work, one B. subtilis strain (WS-1) was successfully isolated from apparently healthy pigs growing with sick cohorts on one E. coli endemic commercial pig farm in Guangdong, China. The B. subtilis strain WS-1 was identified to inhibit the growth of pathogenic E. coli both in vitro and in vivo, indicating its potential application in protecting newborn piglets from diarrhea caused by E. coli infections. The isolation and characterization will help better understand this bacterium, and the strain WS-1 can be further explored as an alternative to antimicrobial drugs to protect human and animal health.

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April 21, 2020

A reference-grade wild soybean genome.

Efficient crop improvement depends on the application of accurate genetic information contained in diverse germplasm resources. Here we report a reference-grade genome of wild soybean accession W05, with a final assembled genome size of 1013.2?Mb and a contig N50 of 3.3?Mb. The analytical power of the W05 genome is demonstrated by several examples. First, we identify an inversion at the locus determining seed coat color during domestication. Second, a translocation event between chromosomes 11 and 13 of some genotypes is shown to interfere with the assignment of QTLs. Third, we find a region containing copy number variations of the Kunitz trypsin inhibitor (KTI) genes. Such findings illustrate the power of this assembly in the analysis of large structural variations in soybean germplasm collections. The wild soybean genome assembly has wide applications in comparative genomic and evolutionary studies, as well as in crop breeding and improvement programs.

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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

Characterization of the Castanopsis carlesii Deadwood Mycobiome by Pacbio Sequencing of the Full-Length Fungal Nuclear Ribosomal Internal Transcribed Spacer (ITS)

Short-read Next Generation Sequencing (NGS) platforms can easily and quickly generate thousands to hundreds of thousands of sequences per sample. However, the limited length of these sequences can cause problems during fungal taxonomic identification. Here we validate the use of Pacbio sequencing, a long-read NGS method, for characterizing the fungal community (mycobiome) of Castanopsis carlesii deadwood. We report the successful use of Pacbio sequencing to generate long-read sequences of the full-length (500 – 780 bp) fungal ITS regions of the Castanopsis carlesii mycobiome. Our results show that the studied deadwood mycobiome is taxonomically and functionally diverse, with an average of 85 fungal OTUs representing five functional groups (animal endosymbionts, endophytes, mycoparasites, plant pathogens, and saprotrophs). Based on relative abundance data, Basidiomycota were the most frequently detected phyla (50% of total sequences), followed by unidentified phyla and Ascomycota. However, based on presence/absence data, the most OTU-rich phyla were Ascomycota (58% of total OTUs, 72 OTUs) followed by Basidiomycota and unidentified phyla. The majority of fungal OTUs were identified as saprotrophs (70% of successfully function-assigned OTUs) followed by plant pathogens. Finally, we used phylogenetic analysis based on the full-length ITS sequences to confirm the species identification of 14/36 OTUs with high bootstrap support (99 – 100%). Based on the numbers of sequence reads obtained per sample, which ranged from 3,047 to 13,463, we conclude that Pacbio sequencing can be a powerful tool for characterizing moderate- and possibly high-complexity fungal communities.

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April 21, 2020

Circular consensus sequencing with long reads.

Long-read sequencing technologies have advantages in genome assembly, structural variant detection and haplotype phasing, but are less suited for single-nucleotide variant (SNV) and insertion/deletion (indel) calling due to the high error rate in comparison with short-read sequencing. Wenger et al., from Pacific Biosciences, optimized the circular consensus sequencing (CCS) protocol to achieve long, high-fidelity reads, in which they selected the SMRTbell library with fractions tightly distributed at 15 kb for high-coverage sequencing.

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April 21, 2020

RNA-seq of HaHV-1-infected abalones reveals a common transcriptional signature of Malacoherpesviruses.

Haliotid herpesvirus-1 (HaHV-1) is the viral agent causative of abalone viral ganglioneuritis, a disease that has severely affected gastropod aquaculture. Although limited, the sequence similarity between HaHV-1 and Ostreid herpesvirus-1 supported the assignment of both viruses to Malacoherpesviridae, a Herpesvirales family distantly related with other viruses. In this study, we reported the first transcriptional data of HaHV-1, obtained from an experimental infection of Haliotis diversicolor supertexta. We also sequenced the genome draft of the Chinese HaHV-1 variant isolated in 2003 (HaHV-1-CN2003) by PacBio technology. Analysis of 13 million reads obtained from 3 RNA samples at 60?hours post injection (hpi) allowed the prediction of 51 new ORFs for a total of 117 viral genes and the identification of 207 variations from the reference genome, consisting in 135 Single Nucleotide Polymorphisms (SNPs) and 72 Insertions or Deletions (InDels). The pairing of genomic and transcriptomic data supported the identification of 60 additional SNPs, representing viral transcriptional variability and preferentially grouped in hotspots. The expression analysis of HaHV-1 ORFs revealed one putative secreted protein, two putative capsid proteins and a possible viral capsid protease as the most expressed genes and demonstrated highly synchronized viral expression patterns of the 3 infected animals at 60?hpi. Quantitative reverse transcription data of 37 viral genes supported the burst of viral transcription at 30 and 60?hpi during the 72?hours of the infection experiment, and allowed the distinction between early and late viral genes.

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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

Getting the Entire Message: Progress in Isoform Sequencing

The advent of second-generation sequencing and its application to RNA sequencing has revolutionized the field of genomics by allowing the quantification of expression of entire genes as well as single TSS, exons and splice sites, RNA-editing sites as well as polyA-sites. However, due to the sequencing of fragments of cDNAs these methods have not given a reliable picture of complete RNA isoforms. Third-generation sequencing has filled this gap and allows end-to-end sequencing of entire RNA/cDNA molecules. This approach to transcriptomics has been a ‘niche’ technology for a couple of years but now is becoming mainstream with many different applications. Here, we review the background and progress made to date in this rapidly growing field. We start by reviewing the progressive realization that alternative splicing is omnipresent. We then focus on long-non-coding RNA isoforms and the distinct combination patterns of exons in non-coding and coding genes. We consider the implications of the recent technologies of direct RNA sequencing and single-cell isoform RNA sequencing. Finally, we discuss the parameters that define the success of long-read RNA sequencing experiments and strategies commonly used to make the most of such data.

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April 21, 2020

The First Highly Contiguous Genome Assembly of Pikeperch (Sander lucioperca), an Emerging Aquaculture Species in Europe

The pikeperch (Sander lucioperca) is a fresh and brackish water Percid fish natively inhabiting the northern hemisphere. This species is emerging as a promising candidate for intensive aquaculture production in Europe. Specific traits like cannibalism, growth rate and meat quality require genomics based understanding, for an optimal husbandry and domestication process. Still, the aquaculture community is lacking an annotated genome sequence to facilitate genome-wide studies on pikeperch. Here, we report the first highly contiguous draft genome assembly of Sander lucioperca. In total, 413 and 66 giga base pairs of DNA sequencing raw data were generated with the Illumina platform and PacBio Sequel System, respectively. The PacBio data were assembled into a final assembly size of ~900 Mb covering 89% of the 1,014 Mb estimated genome size. The draft genome consisted of 1966 contigs ordered into 1,313 scaffolds. The contig and scaffold N50 lengths are 3.0 Mb and 4.9 Mb, respectively. The identified repetitive structures accounted for 39% of the genome. We utilized homologies to other ray-finned fishes, and ab initio gene prediction methods to predict 21,249 protein-coding genes in the Sander lucioperca genome, of which 88% were functionally annotated by either sequence homology or protein domains and signatures search. The assembled genome spans 97.6% and 96.3% of Vertebrate and Actinopterygii single-copy orthologs, respectively. The outstanding mapping rate (99.9%) of genomic PE-reads on the assembly suggests an accurate and nearly complete genome reconstruction. This draft genome sequence is the first genomic resource for this promising aquaculture species. It will provide an impetus for genomic-based breeding studies targeting phenotypic and performance traits of captive pikeperch.

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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

Whole genome sequence and de novo assembly revealed genomic architecture of Indian Mithun (Bos frontalis).

Mithun (Bos frontalis), also called gayal, is an endangered bovine species, under the tribe bovini with 2n?=?58 XX chromosome complements and reared under the tropical rain forests region of India, China, Myanmar, Bhutan and Bangladesh. However, the origin of this species is still disputed and information on its genomic architecture is scanty so far. We trust that availability of its whole genome sequence data and assembly will greatly solve this problem and help to generate many information including phylogenetic status of mithun. Recently, the first genome assembly of gayal, mithun of Chinese origin, was published. However, an improved reference genome assembly would still benefit in understanding genetic variation in mithun populations reared under diverse geographical locations and for building a superior consensus assembly. We, therefore, performed deep sequencing of the genome of an adult female mithun from India, assembled and annotated its genome and performed extensive bioinformatic analyses to produce a superior de novo genome assembly of mithun.We generated ˜300 Gigabyte (Gb) raw reads from whole-genome deep sequencing platforms and assembled the sequence data using a hybrid assembly strategy to create a high quality de novo assembly of mithun with 96% recovered as per BUSCO analysis. The final genome assembly has a total length of 3.0 Gb, contains 5,015 scaffolds with an N50 value of 1?Mb. Repeat sequences constitute around 43.66% of the assembly. The genomic alignments between mithun to cattle showed that their genomes, as expected, are highly conserved. Gene annotation identified 28,044 protein-coding genes presented in mithun genome. The gene orthologous groups of mithun showed a high degree of similarity in comparison with other species, while fewer mithun specific coding sequences were found compared to those in cattle.Here we presented the first de novo draft genome assembly of Indian mithun having better coverage, less fragmented, better annotated, and constitutes a reasonably complete assembly compared to the previously published gayal genome. This comprehensive assembly unravelled the genomic architecture of mithun to a great extent and will provide a reference genome assembly to research community to elucidate the evolutionary history of mithun across its distinct geographical locations.

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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

Single-molecule sequencing detection of N6-methyladenine in microbial reference materials.

The DNA base modification N6-methyladenine (m6A) is involved in many pathways related to the survival of bacteria and their interactions with hosts. Nanopore sequencing offers a new, portable method to detect base modifications. Here, we show that a neural network can improve m6A detection at trained sequence contexts compared to previously published methods using deviations between measured and expected current values as each adenine travels through a pore. The model, implemented as the mCaller software package, can be extended to detect known or confirm suspected methyltransferase target motifs based on predictions of methylation at untrained contexts. We use PacBio, Oxford Nanopore, methylated DNA immunoprecipitation sequencing (MeDIP-seq), and whole-genome bisulfite sequencing data to generate and orthogonally validate methylomes for eight microbial reference species. These well-characterized microbial references can serve as controls in the development and evaluation of future methods for the identification of base modifications from single-molecule sequencing data.

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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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