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April 21, 2020

Long-Read Sequencing Emerging in Medical Genetics

The wide implementation of next-generation sequencing (NGS) technologies has revolutionized the field of medical genetics. However, the short read lengths of currently used sequencing approaches pose a limitation for identification of structural variants, sequencing repetitive regions, phasing alleles and distinguishing highly homologous genomic regions. These limitations may significantly contribute to the diagnostic gap in patients with genetic disorders who have undergone standard NGS, like whole exome or even genome sequencing. Now, the emerging long-read sequencing (LRS) technologies may offer improvements in the characterization of genetic variation and regions that are difficult to assess with the currently prevailing NGS approaches. LRS has so far mainly been used to investigate genetic disorders with previously known or strongly suspected disease loci. While these targeted approaches already show the potential of LRS, it remains to be seen whether LRS technologies can soon enable true whole genome sequencing routinely. Ultimately, this could allow the de novo assembly of individual whole genomes used as a generic test for genetic disorders. In this article, we summarize the current LRS-based research on human genetic disorders and discuss the potential of these technologies to facilitate the next major advancements in medical genetics.

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April 21, 2020

Genomics-driven discovery of a biosynthetic gene cluster required for the synthesis of BII-Rafflesfungin from the fungus Phoma sp. F3723.

Phomafungin is a recently reported broad spectrum antifungal compound but its biosynthetic pathway is unknown. We combed publicly available Phoma genomes but failed to find any putative biosynthetic gene cluster that could account for its biosynthesis.Therefore, we sequenced the genome of one of our Phoma strains (F3723) previously identified as having antifungal activity in a high-throughput screen. We found a biosynthetic gene cluster that was predicted to synthesize a cyclic lipodepsipeptide that differs in the amino acid composition compared to Phomafungin. Antifungal activity guided isolation yielded a new compound, BII-Rafflesfungin, the structure of which was determined.We describe the NRPS-t1PKS cluster ‘BIIRfg’ compatible with the synthesis of the cyclic lipodepsipeptide BII-Rafflesfungin [HMHDA-L-Ala-L-Glu-L-Asn-L-Ser-L-Ser-D-Ser-D-allo-Thr-Gly]. We report new Stachelhaus codes for Ala, Glu, Asn, Ser, Thr, and Gly. We propose a mechanism for BII-Rafflesfungin biosynthesis, which involves the formation of the lipid part by BIIRfg_PKS followed by activation and transfer of the lipid chain by a predicted AMP-ligase on to the first PCP domain of the BIIRfg_NRPS gene.

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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

Divergent evolutionary trajectories following speciation in two ectoparasitic honey bee mites.

Multispecies host-parasite evolution is common, but how parasites evolve after speciating remains poorly understood. Shared evolutionary history and physiology may propel species along similar evolutionary trajectories whereas pursuing different strategies can reduce competition. We test these scenarios in the economically important association between honey bees and ectoparasitic mites by sequencing the genomes of the sister mite species Varroa destructor and Varroa jacobsoni. These genomes were closely related, with 99.7% sequence identity. Among the 9,628 orthologous genes, 4.8% showed signs of positive selection in at least one species. Divergent selective trajectories were discovered in conserved chemosensory gene families (IGR, SNMP), and Halloween genes (CYP) involved in moulting and reproduction. However, there was little overlap in these gene sets and associated GO terms, indicating different selective regimes operating on each of the parasites. Based on our findings, we suggest that species-specific strategies may be needed to combat evolving parasite communities. © The Author(s) 2019.

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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

Analysis of genetic diversity of Xanthomonas oryzae pv. oryzae populations in Taiwan.

Rice bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is a major rice disease. In Taiwan, the tropical indica type of Oryza sativa originally grown in this area is mix-cultivated with the temperate japonica type of O. sativa, and this might have led to adaptive changes of both rice host and Xoo isolates. In order to better understand how Xoo adapts to this unique environment, we collected and analyzed fifty-one Xoo isolates in Taiwan. Three different genetic marker systems consistently identified five groups. Among these groups, two of them had unique sequences in the last acquired ten spacers in the clustered regularly interspaced short palindromic repeats (CRISPR) region, and the other two had sequences that were similar to the Japanese isolate MAFF311018 and the Philippines isolate PXO563, respectively. The genomes of two Taiwanese isolates with unique CRISPR sequence features, XF89b and XM9, were further completely sequenced. Comparison of the genome sequences suggested that XF89b is phylogenetically close to MAFF311018, and XM9 is close to PXO563. Here, documentation of the diversity of groups of Xoo in Taiwan provides evidence of the populations from different sources and hitherto missing information regarding distribution of Xoo populations in East Asia.

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April 21, 2020

The transcriptome of Darwin’s bark spider silk glands predicts proteins contributing to dragline silk toughness.

Darwin’s bark spider (Caerostris darwini) produces giant orb webs from dragline silk that can be twice as tough as other silks, making it the toughest biological material. This extreme toughness comes from increased extensibility relative to other draglines. We show C. darwini dragline-producing major ampullate (MA) glands highly express a novel silk gene transcript (MaSp4) encoding a protein that diverges markedly from closely related proteins and contains abundant proline, known to confer silk extensibility, in a unique GPGPQ amino acid motif. This suggests C. darwini evolved distinct proteins that may have increased its dragline’s toughness, enabling giant webs. Caerostris darwini’s MA spinning ducts also appear unusually long, potentially facilitating alignment of silk proteins into extremely tough fibers. Thus, a suite of novel traits from the level of genes to spinning physiology to silk biomechanics are associated with the unique ecology of Darwin’s bark spider, presenting innovative designs for engineering biomaterials.

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April 21, 2020

A hybrid de novo genome assembly of the honeybee, Apis mellifera, with chromosome-length scaffolds.

The ability to generate long sequencing reads and access long-range linkage information is revolutionizing the quality and completeness of genome assemblies. Here we use a hybrid approach that combines data from four genome sequencing and mapping technologies to generate a new genome assembly of the honeybee Apis mellifera. We first generated contigs based on PacBio sequencing libraries, which were then merged with linked-read 10x Chromium data followed by scaffolding using a BioNano optical genome map and a Hi-C chromatin interaction map, complemented by a genetic linkage map.Each of the assembly steps reduced the number of gaps and incorporated a substantial amount of additional sequence into scaffolds. The new assembly (Amel_HAv3) is significantly more contiguous and complete than the previous one (Amel_4.5), based mainly on Sanger sequencing reads. N50 of contigs is 120-fold higher (5.381 Mbp compared to 0.053 Mbp) and we anchor >?98% of the sequence to chromosomes. All of the 16 chromosomes are represented as single scaffolds with an average of three sequence gaps per chromosome. The improvements are largely due to the inclusion of repetitive sequence that was unplaced in previous assemblies. In particular, our assembly is highly contiguous across centromeres and telomeres and includes hundreds of AvaI and AluI repeats associated with these features.The improved assembly will be of utility for refining gene models, studying genome function, mapping functional genetic variation, identification of structural variants, and comparative genomics.

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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

Long-read sequencing reveals a 4.4 kb tandem repeat region in the mitogenome of Echinococcus granulosus (sensu stricto) genotype G1.

Echinococcus tapeworms cause a severe helminthic zoonosis called echinococcosis. The genus comprises various species and genotypes, of which E. granulosus (sensu stricto) represents a significant global public health and socioeconomic burden. Mitochondrial (mt) genomes have provided useful genetic markers to explore the nature and extent of genetic diversity within Echinococcus and have underpinned phylogenetic and population structure analyses of this genus. Our recent work indicated a sequence gap (>?1 kb) in the mt genomes of E. granulosus genotype G1, which could not be determined by PCR-based Sanger sequencing. The aim of the present study was to define the complete mt genome, irrespective of structural complexities, using a long-read sequencing method.We extracted high molecular weight genomic DNA from protoscoleces from a single cyst of E. granulosus genotype G1 from a sheep from Australia using a conventional method and sequenced it using PacBio Sequel (long-read) technology, complemented by BGISEQ-500 short-read sequencing. Sequence data obtained were assembled using a recently-developed workflow.We assembled a complete mt genome sequence of 17,675 bp, which is >?4 kb larger than the complete mt genomes known for E. granulosus genotype G1. This assembly includes a previously-elusive tandem repeat region, which is 4417 bp long and consists of ten near-identical 441-445 bp repeat units, each harbouring a 184 bp non-coding region and adjacent regions. We also identified a short non-coding region of 183 bp, which includes an inverted repeat.We report what we consider to be the first complete mt genome of E. granulosus genotype G1 and characterise all repeat regions in this genome. The numbers, sizes, sequences and functions of tandem repeat regions remain to be studied in different isolates of genotype G1 and in other genotypes and species. The discovery of such ‘new’ repeat elements in the mt genome of genotype G1 by PacBio sequencing raises a question about the completeness of some published genomes of taeniid cestodes assembled from conventional or short-read sequence datasets. This study shows that long-read sequencing readily overcomes the challenges of assembling repeat elements to achieve improved genomes.

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April 21, 2020

Genome of lethal Lepiota venenata and insights into the evolution of toxin-biosynthetic genes.

Genomes of lethal Amanita and Galerina mushrooms have gradually become available in the past ten years; in contrast the other known amanitin-producing genus, Lepiota, is still vacant in this aspect. A fatal mushroom poisoning case in China has led to acquisition of fresh L. venenata fruiting bodies, based on which a draft genome was obtained through PacBio and Illumina sequencing platforms. Toxin-biosynthetic MSDIN family and Porlyl oligopeptidase B (POPB) genes were mined from the genome and used for phylogenetic and statistical studies to gain insights into the evolution of the biosynthetic pathway.The analysis of the genome data illustrated that only one MSDIN, named LvAMA1, exits in the genome, along with a POPB gene. No POPA homolog was identified by direct homology searching, however, one additional POP gene, named LvPOPC, was cloned and the gene structure determined. Similar to ApAMA1 in A. phalloides and GmAMA1 in G. marginata, LvAMA1 directly encodes a-amanitin. The two toxin genes were mapped to the draft genome, and the structures analyzed. Furthermore, phylogenetic and statistical analyses were conducted to study the evolution history of the POPB genes. Compared to our previous report, the phylogenetic trees unambiguously showed that a monophyletic POPB lineage clearly conflicted with the species phylogeny. In contrast, phylogeny of POPA genes resembled the species phylogeny. Topology and divergence tests showed that the POPB lineage was robust and these genes exhibited significantly shorter genetic distances than those of the house-keeping rbp2, a characteristic feature of genes with horizontal gene transfer (HGT) background. Consistently, same scenario applied to the only MSDIN, LvAMA1, in the genome.To the best of our knowledge, this is the first reported genome of Lepiota. The analyses of the toxin genes indicate that the cyclic peptides are synthesized through a ribosomal mechanism. The toxin genes, LvAMA1 and LvPOPB, are not in the vicinity of each other. Phylogenetic and evolutionary studies suggest that HGT is the underlining cause for the occurrence of POPB and MSDIN in Amanita, Galerina and Lepiota, which are allocated in three distantly-related families.

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April 21, 2020

Comparative genomics reveals structural and functional features specific to the genome of a foodborne Escherichia coli O157:H7.

Escherichia coli O157:H7 (O157) has been linked to numerous foodborne disease outbreaks. The ability to rapidly sequence and analyze genomes is important for understanding epidemiology, virulence, survival, and evolution of outbreak strains. In the current study, we performed comparative genomics to determine structural and functional features of the genome of a foodborne O157 isolate NADC 6564 and infer its evolutionary relationship to other O157 strains.The chromosome of NADC 6564 contained 5466?kb compared to reference strains Sakai (5498?kb) and EDL933 (5547?kb) and shared 41 of its 43 Linear Conserved Blocks (LCB) with the reference strains. However, 18 of 41 LCB had inverse orientation in NADC 6564 compared to the reference strains. NADC 6564 shared 18 of 19 bacteriophages with reference strains except that the chromosomal positioning of some of the phages differed among these strains. The additional phage (P19) of NADC 6564 was located on a 39-kb insertion element (IE) encoding several hypothetical proteins, an integrase, transposases, transcriptional regulators, an adhesin, and a phosphoethanolamine transferase (PEA). The complete homologs of the 39-kb?IE were found in E. coli PCN061 of porcine origin. The IE-encoded PEA showed low homology (32-33%) to four other PEA in NADC 6564 and PEA linked to mobilizable colistin resistance in E. coli but was highly homologous (95%) to a PEA of uropathogenic, avian pathogenic, and enteroaggregative E. coli. NADC 6564 showed slightly higher minimum inhibitory concentration of colistin compared to the reference strains. The 39-kb?IE also contained dndBCDE and dptFGH operons encoding DNA S-modification and a restriction pathway, linked to oxidative stress tolerance and self-defense against foreign DNA, respectively. Evolutionary tree analysis grouped NADC 6564 with lineage I O157 strains.These results indicated that differential phage counts and different chromosomal positioning of many bacteriophages and genomic islands might have resulted in recombination events causing altered chromosomal organization in NADC 6564. Evolutionary analysis grouped NADC 6564 with lineage I strains and suggested its earlier divergence from these strains. The ability to perform S-DNA modification might affect tolerance of NADC 6564 to various stressors.

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April 21, 2020

A First Study of the Virulence Potential of a Bacillus subtilis Isolate From Deep-Sea Hydrothermal Vent.

Bacillus subtilis is the best studied Gram-positive bacterium, primarily as a model of cell differentiation and industrial exploitation. To date, little is known about the virulence of B. subtilis. In this study, we examined the virulence potential of a B. subtilis strain (G7) isolated from the Iheya North hydrothermal field of Okinawa Trough. G7 is aerobic, motile, endospore-forming, and requires NaCl for growth. The genome of G7 is composed of one circular chromosome of 4,216,133 base pairs with an average GC content of 43.72%. G7 contains 4,416 coding genes, 27.5% of which could not be annotated, and the remaining 72.5% were annotated with known or predicted functions in 25 different COG categories. Ten sets of 23S, 5S, and 16S ribosomal RNA operons, 86 tRNA and 14 sRNA genes, 50 tandem repeats, 41 mini-satellites, one microsatellite, and 42 transposons were identified in G7. Comparing to the genome of the B. subtilis wild type strain NCIB 3610T, G7 genome contains many genomic translocations, inversions, and insertions, and twice the amount of genomic Islands (GIs), with 42.5% of GI genes encoding hypothetical proteins. G7 possesses abundant putative virulence genes associated with adhesion, invasion, dissemination, anti-phagocytosis, and intracellular survival. Experimental studies showed that G7 was able to cause mortality in fish and mice following intramuscular/intraperitoneal injection, resist the killing effect of serum complement, and replicate in mouse macrophages and fish peripheral blood leukocytes. Taken together, our study indicates that G7 is a B. subtilis isolate with unique genetic features and can be lethal to vertebrate animals once being introduced into the animals by artificial means. These results provide the first insight into the potential harmfulness of deep-sea B. subtilis.

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April 21, 2020

De novo transcriptome assembly of the cubomedusa Tripedalia cystophora, including the analysis of a set of genes involved in peptidergic neurotransmission.

The phyla Cnidaria, Placozoa, Ctenophora, and Porifera emerged before the split of proto- and deuterostome animals, about 600 million years ago. These early metazoans are interesting, because they can give us important information on the evolution of various tissues and organs, such as eyes and the nervous system. Generally, cnidarians have simple nervous systems, which use neuropeptides for their neurotransmission, but some cnidarian medusae belonging to the class Cubozoa (box jellyfishes) have advanced image-forming eyes, probably associated with a complex innervation. Here, we describe a new transcriptome database from the cubomedusa Tripedalia cystophora.Based on the combined use of the Illumina and PacBio sequencing technologies, we produced a highly contiguous transcriptome database from T. cystophora. We then developed a software program to discover neuropeptide preprohormones in this database. This script enabled us to annotate seven novel T. cystophora neuropeptide preprohormone cDNAs: One coding for 19 copies of a peptide with the structure pQWLRGRFamide; one coding for six copies of a different RFamide peptide; one coding for six copies of pQPPGVWamide; one coding for eight different neuropeptide copies with the C-terminal LWamide sequence; one coding for thirteen copies of a peptide with the RPRAamide C-terminus; one coding for four copies of a peptide with the C-terminal GRYamide sequence; and one coding for seven copies of a cyclic peptide, of which the most frequent one has the sequence CTGQMCWFRamide. We could also identify orthologs of these seven preprohormones in the cubozoans Alatina alata, Carybdea xaymacana, Chironex fleckeri, and Chiropsalmus quadrumanus. Furthermore, using TBLASTN screening, we could annotate four bursicon-like glycoprotein hormone subunits, five opsins, and 52 other family-A G protein-coupled receptors (GPCRs), which also included two leucine-rich repeats containing G protein-coupled receptors (LGRs) in T. cystophora. The two LGRs are potential receptors for the glycoprotein hormones, while the other GPCRs are candidate receptors for the above-mentioned neuropeptides.By combining Illumina and PacBio sequencing technologies, we have produced a new high-quality de novo transcriptome assembly from T. cystophora that should be a valuable resource for identifying the neuronal components that are involved in vision and other behaviors in cubomedusae.

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April 21, 2020

Comparative genomics and pathogenicity potential of members of the Pseudomonas syringae species complex on Prunus spp.

Diseases on Prunus spp. have been associated with a large number of phylogenetically different pathovars and species within the P. syringae species complex. Despite their economic significance, there is a severe lack of genomic information of these pathogens. The high phylogenetic diversity observed within strains causing disease on Prunus spp. in nature, raised the question whether other strains or species within the P. syringae species complex were potentially pathogenic on Prunus spp.To gain insight into the genomic potential of adaptation and virulence in Prunus spp., a total of twelve de novo whole genome sequences of P. syringae pathovars and species found in association with diseases on cherry (sweet, sour and ornamental-cherry) and peach were sequenced. Strains sequenced in this study covered three phylogroups and four clades. These strains were screened in vitro for pathogenicity on Prunus spp. together with additional genome sequenced strains thus covering nine out of thirteen of the currently defined P. syringae phylogroups. Pathogenicity tests revealed that most of the strains caused symptoms in vitro and no obvious link was found between presence of known virulence factors and the observed pathogenicity pattern based on comparative genomics. Non-pathogenic strains were displaying a two to three times higher generation time when grown in rich medium.In this study, the first set of complete genomes of cherry associated P. syringae strains as well as the draft genome of the quarantine peach pathogen P. syringae pv. persicae were generated. The obtained genomic data were matched with phenotypic data in order to determine factors related to pathogenicity to Prunus spp. Results of this study suggest that the inability to cause disease on Prunus spp. in vitro is not the result of host specialization but rather linked to metabolic impairments of individual strains.

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