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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.


April 21, 2020  |  

Comprehensive characterization of T-DNA integration induced chromosomal rearrangement in a birch T-DNA mutant.

Integration of T-DNA into plant genomes via Agrobacterium may interrupt gene structure and generate numerous mutants. The T-DNA caused mutants are valuable materials for understanding T-DNA integration model in plant research. T-DNA integration in plants is complex and still largely unknown. In this work, we reported that multiple T-DNA fragments caused chromosomal translocation and deletion in a birch (Betula platyphylla × B. pendula) T-DNA mutant yl.We performed PacBio genome resequencing for yl and the result revealed that two ends of a T-DNA can be integrated into plant genome independently because the two ends can be linked to different chromosomes and cause chromosomal translocation. We also found that these T-DNA were connected into tandem fragment regardless of direction before integrating into plant genome. In addition, the integration of T-DNA in yl genome also caused several chromosomal fragments deletion. We then summarized three cases for T-DNA integration model in the yl genome. (1) A T-DNA fragment is linked to the two ends of a double-stranded break (DSB); (2) Only one end of a T-DNA fragment is linked to a DSB; (3) A T-DNA fragment is linked to the ends of different DSBs. All the observations in the yl genome supported the DSB repair model.In this study, we showed a comprehensive genome analysis of a T-DNA mutant and provide a new insight into T-DNA integration in plants. These findings would be helpful for the analysis of T-DNA mutants with special phenotypes.


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.


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.


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.


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).


April 21, 2020  |  

FGMP: assessing fungal genome completeness

Background: Inexpensive high-throughput DNA sequencing has democratized access to genetic information for most organisms so that research utilizing a genome or transcriptome of an organism is not limited to model systems. However, the quality of the assemblies of sampled genomes can vary greatly which hampers utility for comparisons and meaningful interpretation. The uncertainty of the completeness of a given genome sequence can limit feasibility of asserting patterns of high rates of gene loss reported in many lineages. Results: We propose a computational framework and sequence resource for assessing completeness of fungal genomes called FGMP (Fungal Genome Mapping Project). Our approach is based on evolutionary conserved sets of proteins and DNA elements and is applicable to various types of genomic data. We present a comparison of FGMP and state-of-the-art methods for genome completeness assessment utilizing 246 genome assemblies of fungi. We discuss genome assembly improvements/degradations in 57 cases where assemblies have been updated, as recorded by NCBI assembly archive. Conclusion: FGMP is an accurate tool for quantifying level of completion from fungal genomic data. It is particularly useful for non-model organisms without reference genomes and can be used directly on unassembled reads, which can help reducing genome sequencing costs.


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.


April 21, 2020  |  

Metaepigenomic analysis reveals the unexplored diversity of DNA methylation in an environmental prokaryotic community.

DNA methylation plays important roles in prokaryotes, and their genomic landscapes-prokaryotic epigenomes-have recently begun to be disclosed. However, our knowledge of prokaryotic methylation systems is focused on those of culturable microbes, which are rare in nature. Here, we used single-molecule real-time and circular consensus sequencing techniques to reveal the ‘metaepigenomes’ of a microbial community in the largest lake in Japan, Lake Biwa. We reconstructed 19 draft genomes from diverse bacterial and archaeal groups, most of which are yet to be cultured. The analysis of DNA chemical modifications in those genomes revealed 22 methylated motifs, nine of which were novel. We identified methyltransferase genes likely responsible for methylation of the novel motifs, and confirmed the catalytic specificities of four of them via transformation experiments using synthetic genes. Our study highlights metaepigenomics as a powerful approach for identification of the vast unexplored variety of prokaryotic DNA methylation systems in nature.


April 21, 2020  |  

Assignment of virus and antimicrobial resistance genes to microbial hosts in a complex microbial community by combined long-read assembly and proximity ligation.

We describe a method that adds long-read sequencing to a mix of technologies used to assemble a highly complex cattle rumen microbial community, and provide a comparison to short read-based methods. Long-read alignments and Hi-C linkage between contigs support the identification of 188 novel virus-host associations and the determination of phage life cycle states in the rumen microbial community. The long-read assembly also identifies 94 antimicrobial resistance genes, compared to only seven alleles in the short-read assembly. We demonstrate novel techniques that work synergistically to improve characterization of biological features in a highly complex rumen microbial community.


April 21, 2020  |  

Long-read based de novo assembly of low-complexity metagenome samples results in finished genomes and reveals insights into strain diversity and an active phage system.

Complete and contiguous genome assemblies greatly improve the quality of subsequent systems-wide functional profiling studies and the ability to gain novel biological insights. While a de novo genome assembly of an isolated bacterial strain is in most cases straightforward, more informative data about co-existing bacteria as well as synergistic and antagonistic effects can be obtained from a direct analysis of microbial communities. However, the complexity of metagenomic samples represents a major challenge. While third generation sequencing technologies have been suggested to enable finished metagenome-assembled genomes, to our knowledge, the complete genome assembly of all dominant strains in a microbiome sample has not been demonstrated. Natural whey starter cultures (NWCs) are used in cheese production and represent low-complexity microbiomes. Previous studies of Swiss Gruyère and selected Italian hard cheeses, mostly based on amplicon metagenomics, concurred that three species generally pre-dominate: Streptococcus thermophilus, Lactobacillus helveticus and Lactobacillus delbrueckii.Two NWCs from Swiss Gruyère producers were subjected to whole metagenome shotgun sequencing using the Pacific Biosciences Sequel and Illumina MiSeq platforms. In addition, longer Oxford Nanopore Technologies MinION reads had to be generated for one to resolve repeat regions. Thereby, we achieved the complete assembly of all dominant bacterial genomes from these low-complexity NWCs, which was corroborated by a 16S rRNA amplicon survey. Moreover, two distinct L. helveticus strains were successfully co-assembled from the same sample. Besides bacterial chromosomes, we could also assemble several bacterial plasmids and phages and a corresponding prophage. Biologically relevant insights were uncovered by linking the plasmids and phages to their respective host genomes using DNA methylation motifs on the plasmids and by matching prokaryotic CRISPR spacers with the corresponding protospacers on the phages. These results could only be achieved by employing long-read sequencing data able to span intragenomic as well as intergenomic repeats.Here, we demonstrate the feasibility of complete de novo genome assembly of all dominant strains from low-complexity NWCs based on whole metagenomics shotgun sequencing data. This allowed to gain novel biological insights and is a fundamental basis for subsequent systems-wide omics analyses, functional profiling and phenotype to genotype analysis of specific microbial communities.


April 21, 2020  |  

Cichorium intybus L.?×?Cicerbita alpina Walbr.: doubled haploid chicory induction and CENH3 characterization

Intergeneric hybridization between industrial chicory (Cichorium intybus L.) and Cicerbita alpina Walbr. induces interspecific hybrids and haploid chicory plants after in vitro embryo rescue. The protocol yielded haploids in 5 out of 12 cultivars pollinated; altogether 18 haploids were regenerated from 2836 embryos, with a maximum efficiency of 1.96% haploids per cross. Obtained haploids were chromosome doubled with mitosis inhibitors trifluralin and oryzalin; exposure to 0.05 g L-1 oryzalin during one week was the most efficient treatment to regenerate doubled haploids. Inbreeding effects in vitro were limited, but the ploidy level affects morphology. Transcriptome sequencing revealed two unique copies of CENH3 in Cicerbita alpina Walbr. Comparison of CENH3.1 protein sequences of Cicerbita and Cichorium obtained through transcriptome and whole shotgun genome sequencing revealed two amino-acid substitutions at critical residues of the histone fold domain. These particular changes cause chromosome elimination and reduced centromere loading in several other species and might indicate a CENH3-dependent mechanism causing chromosome elimination of parental chromosomes during Cichorium?×?Cicerbita intergeneric hybridization. Our results provide insights in chromosome elimination and might increase the efficiency of haploid induction in Cichorium.


April 21, 2020  |  

Chromosome assembly of Collichthys lucidus, a fish of Sciaenidae with a multiple sex chromosome system.

Collichthys lucidus (C. lucidus) is a commercially important marine fish species distributed in coastal regions of East Asia with the X1X1X2X2/X1X2Y multiple sex chromosome system. The karyotype for female C. lucidus is 2n?=?48, while 2n?=?47 for male ones. Therefore, C. lucidus is also an excellent model to investigate teleost sex-determination and sex chromosome evolution. We reported the first chromosome genome assembly of C. lucidus using Illumina short-read, PacBio long-read sequencing and Hi-C technology. An 877?Mb genome was obtained with a contig and scaffold N50 of 1.1?Mb and 35.9?Mb, respectively. More than 97% BUSCOs genes were identified in the C. lucidus genome and 28,602 genes were annotated. We identified potential sex-determination genes along chromosomes and found that the chromosome 1 might be involved in the formation of Y specific metacentric chromosome. The first C. lucidus chromosome-level reference genome lays a solid foundation for the following population genetics study, functional gene mapping of important economic traits, sex-determination and sex chromosome evolution studies for Sciaenidae and teleosts.


April 21, 2020  |  

The wild sweetpotato (Ipomoea trifida) genome provides insights into storage root development.

Sweetpotato (Ipomoea batatas (L.) Lam.) is the seventh most important crop in the world and is mainly cultivated for its underground storage root (SR). The genetic studies of this species have been hindered by a lack of high-quality reference sequence due to its complex genome structure. Diploid Ipomoea trifida is the closest relative and putative progenitor of sweetpotato, which is considered a model species for sweetpotato, including genetic, cytological, and physiological analyses.Here, we generated the chromosome-scale genome sequence of SR-forming diploid I. trifida var. Y22 with high heterozygosity (2.20%). Although the chromosome-based synteny analysis revealed that the I. trifida shared conserved karyotype with Ipomoea nil after the separation, I. trifida had a much smaller genome than I. nil due to more efficient eliminations of LTR-retrotransposons and lack of species-specific amplification bursts of LTR-RTs. A comparison with four non-SR-forming species showed that the evolution of the beta-amylase gene family may be related to SR formation. We further investigated the relationship of the key gene BMY11 (with identity 47.12% to beta-amylase 1) with this important agronomic trait by both gene expression profiling and quantitative trait locus (QTL) mapping. And combining SR morphology and structure, gene expression profiling and qPCR results, we deduced that the products of the activity of BMY11 in splitting starch granules and be recycled to synthesize larger granules, contributing to starch accumulation and SR swelling. Moreover, we found the expression pattern of BMY11, sporamin proteins and the key genes involved in carbohydrate metabolism and stele lignification were similar to that of sweetpotato during the SR development.We constructed the high-quality genome reference of the highly heterozygous I. trifida through a combined approach and this genome enables a better resolution of the genomics feature and genome evolutions of this species. Sweetpotato SR development genes can be identified in I. trifida and these genes perform similar functions and patterns, showed that the diploid I. trifida var. Y22 with typical SR could be considered an ideal model for the studies of sweetpotato SR development.


April 21, 2020  |  

Characterization of a male specific region containing a candidate sex determining gene in Atlantic cod.

The genetic mechanisms determining sex in teleost fishes are highly variable and the master sex determining gene has only been identified in few species. Here we characterize a male-specific region of 9?kb on linkage group 11 in Atlantic cod (Gadus morhua) harboring a single gene named zkY for zinc knuckle on the Y chromosome. Diagnostic PCR test of phenotypically sexed males and females confirm the sex-specific nature of the Y-sequence. We identified twelve highly similar autosomal gene copies of zkY, of which eight code for proteins containing the zinc knuckle motif. 3D modeling suggests that the amino acid changes observed in six copies might influence the putative RNA-binding specificity. Cod zkY and the autosomal proteins zk1 and zk2 possess an identical zinc knuckle structure, but only the Y-specific gene zkY was expressed at high levels in the developing larvae before the onset of sex differentiation. Collectively these data suggest zkY as a candidate master masculinization gene in Atlantic cod. PCR amplification of Y-sequences in Arctic cod (Arctogadus glacialis) and Greenland cod (Gadus macrocephalus ogac) suggests that the male-specific region emerged in codfishes more than 7.5 million years ago.


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