Menu
September 22, 2019

Two novel lncRNAs discovered in human mitochondrial DNA using PacBio full-length transcriptome data.

In this study, we established a general framework to use PacBio full-length transcriptome sequencing for the investigation of mitochondrial RNAs. As a result, we produced the first full-length human mitochondrial transcriptome using public PacBio data and characterized the human mitochondrial genome with more comprehensive and accurate information. Other results included determination of the H-strand primary transcript, identification of the ND5/ND6AS/tRNAGluAS transcript, discovery of palindrome small RNAs (psRNAs) and construction of the “mitochondrial cleavage” model, etc. These results reported for the first time in this study fundamentally changed annotations of human mitochondrial genome and enriched knowledge in the field of animal mitochondrial studies. The most important finding was two novel long non-coding RNAs (lncRNAs) of MDL1 and MDL1AS exist ubiquitously in animal mitochondrial genomes. Copyright © 2017. Published by Elsevier B.V.


September 22, 2019

Accurate characterization of the IFITM locus using MiSeq and PacBio sequencing shows genetic variation in Galliformes.

Interferon inducible transmembrane (IFITM) proteins are effectors of the immune system widely characterized for their role in restricting infection by diverse enveloped and non-enveloped viruses. The chicken IFITM (chIFITM) genes are clustered on chromosome 5 and to date four genes have been annotated, namely chIFITM1, chIFITM3, chIFITM5 and chIFITM10. However, due to poor assembly of this locus in the Gallus Gallus v4 genome, accurate characterization has so far proven problematic. Recently, a new chicken reference genome assembly Gallus Gallus v5 was generated using Sanger, 454, Illumina and PacBio sequencing technologies identifying considerable differences in the chIFITM locus over the previous genome releases.We re-sequenced the locus using both Illumina MiSeq and PacBio RS II sequencing technologies and we mapped RNA-seq data from the European Nucleotide Archive (ENA) to this finalized chIFITM locus. Using SureSelect probes capture probes designed to the finalized chIFITM locus, we sequenced the locus of a different chicken breed, namely a White Leghorn, and a turkey.We confirmed the Gallus Gallus v5 consensus except for two insertions of 5 and 1 base pair within the chIFITM3 and B4GALNT4 genes, respectively, and a single base pair deletion within the B4GALNT4 gene. The pull down revealed a single amino acid substitution of A63V in the CIL domain of IFITM2 compared to Red Jungle fowl and 13, 13 and 11 differences between IFITM1, 2 and 3 of chickens and turkeys, respectively. RNA-seq shows chIFITM2 and chIFITM3 expression in numerous tissue types of different chicken breeds and avian cell lines, while the expression of the putative chIFITM1 is limited to the testis, caecum and ileum tissues.Locus resequencing using these capture probes and RNA-seq based expression analysis will allow the further characterization of genetic diversity within Galliformes.


September 22, 2019

The discovered chimeric protein plays the cohesive role to maintain scallop byssal root structural integrity.

Adhesion is essential for many marine sessile organisms. Unraveling the compositions and assembly of marine bioadheisves is the fundamental to understand their physiological roles. Despite the remarkable diversity of animal bioadhesion, our understanding of this biological process remains limited to only a few animal lineages, leaving the majority of lineages remain enigmatic. Our previous study demonstrated that scallop byssus had distinct protein composition and unusual assembly mechanism apart from mussels. Here a novel protein (Sbp9) was discovered from the key part of the byssus (byssal root), which contains two Calcium Binding Domain (CBD) and 49 tandem Epidermal Growth Factor-Like (EGFL) domain repeats. Modular architecture of Sbp9 represents a novel chimeric gene family resulting from a gene fusion event through the acquisition of CBD2 domain by tenascin like (TNL) gene from Na+/Ca2+ exchanger 1 (NCX1) gene. Finally, free thiols are present in Sbp9 and the results of a rescue assay indicated that Sbp9 likely plays the cohesive role for byssal root integrity. This study not only aids our understanding of byssus assembly but will also inspire biomimetic material design.


September 22, 2019

Analyses of alternative polyadenylation: from old school biochemistry to high-throughput technologies.

Alternations in usage of polyadenylation sites during transcription termination yield transcript isoforms from a gene. Recent findings of transcriptome-wide alternative polyadenylation (APA) as a molecular response to changes in biology position APA not only as a molecular event of early transcriptional termination but also as a cellular regulatory step affecting various biological pathways. With the development of high-throughput profiling technologies at a single nucleotide level and their applications targeted to the 3′-end of mRNAs, dynamics in the landscape of mRNA 3′-end is measureable at a global scale. In this review, methods and technologies that have been adopted to study APA events are discussed. In addition, various bioinformatics algorithms for APA isoform analysis using publicly available RNA-seq datasets are introduced. [BMB Reports 2017; 50(4): 201-207].


September 22, 2019

The full transcription map of mouse papillomavirus type 1 (MmuPV1) in mouse wart tissues.

Mouse papillomavirus type 1 (MmuPV1) provides, for the first time, the opportunity to study infection and pathogenesis of papillomaviruses in the context of laboratory mice. In this report, we define the transcriptome of MmuPV1 genome present in papillomas arising in experimentally infected mice using a combination of RNA-seq, PacBio Iso-seq, 5′ RACE, 3′ RACE, primer-walking RT-PCR, RNase protection, Northern blot and in situ hybridization analyses. We demonstrate that the MmuPV1 genome is transcribed unidirectionally from five major promoters (P) or transcription start sites (TSS) and polyadenylates its transcripts at two major polyadenylation (pA) sites. We designate the P7503, P360 and P859 as “early” promoters because they give rise to transcripts mostly utilizing the polyadenylation signal at nt 3844 and therefore can only encode early genes, and P7107 and P533 as “late” promoters because they give rise to transcripts utilizing polyadenylation signals at either nt 3844 or nt 7047, the latter being able to encode late, capsid proteins. MmuPV1 genome contains five splice donor sites and three acceptor sites that produce thirty-six RNA isoforms deduced to express seven predicted early gene products (E6, E7, E1, E1^M1, E1^M2, E2 and E8^E2) and three predicted late gene products (E1^E4, L2 and L1). The majority of the viral early transcripts are spliced once from nt 757 to 3139, while viral late transcripts, which are predicted to encode L1, are spliced twice, first from nt 7243 to either nt 3139 (P7107) or nt 757 to 3139 (P533) and second from nt 3431 to nt 5372. Thirteen of these viral transcripts were detectable by Northern blot analysis, with the P533-derived late E1^E4 transcripts being the most abundant. The late transcripts could be detected in highly differentiated keratinocytes of MmuPV1-infected tissues as early as ten days after MmuPV1 inoculation and correlated with detection of L1 protein and viral DNA amplification. In mature warts, detection of L1 was also found in more poorly differentiated cells, as previously reported. Subclinical infections were also observed. The comprehensive transcription map of MmuPV1 generated in this study provides further evidence that MmuPV1 is similar to high-risk cutaneous beta human papillomaviruses. The knowledge revealed will facilitate the use of MmuPV1 as an animal virus model for understanding of human papillomavirus gene expression, pathogenesis and immunology.


September 22, 2019

Whole genome sequencing of “Faecalibaculum rodentium” ALO17, isolated from C57BL/6J laboratory mouse feces.

Intestinal microorganisms affect host physiology, including ageing. Given the difficulty in controlling for human studies of the gut microbiome, mouse models provide an alternative avenue to study such relationships. In this study, we report on the complete genome of “Faecalibaculum rodentium” ALO17, a bacterium that was isolated from the faeces of a 9-month-old female C57BL/6J mouse. This strain will be utilized in future in vivo studies detailing the relationships between the gut microbiome and ageing.The whole genome sequence of “F. rodentium” ALO17 was obtained using single-molecule, real-time (SMRT) technique on a PacBio instrument. The assembled genome consisted of 2,542,486 base pairs of double-stranded DNA with a GC content of 54.0 % and no plasmids. The genome was predicted to contain 2794 open reading frames, 55 tRNA genes, and 38 rRNA genes. The 16S rRNA gene of ALO17 was 86.9 % similar to that of Allobaculum stercoricanis DSM 13633(T), and the average overall nucleotide identity between strains ALO17 and DSM 13633(T) was 66.8 %. After confirming the phylogenetic relationship between “F. rodentium” ALO17 and A. stercoricanis DSM 13633(T), their whole genome sequences were compared, revealing that “F. rodentium” ALO17 contains more fermentation-related genes than A. stercoricanis DSM 13633(T). Furthermore, “F. rodentium” ALO17 produces higher levels of lactic acid than A. stercoricanis DSM 13633(T) as determined by high-performance liquid chromatography.The availability of the “F. rodentium” ALO17 whole genome sequence will enhance studies concerning the gut microbiota and host physiology, especially when investigating the molecular relationships between gut microbiota and ageing.


September 22, 2019

Long-read sequencing of human cytomegalovirus transcriptome reveals RNA isoforms carrying distinct coding potentials.

The human cytomegalovirus (HCMV) is a ubiquitous, human pathogenic herpesvirus. The complete viral genome is transcriptionally active during infection; however, a large part of its transcriptome has yet to be annotated. In this work, we applied the amplified isoform sequencing technique from Pacific Biosciences to characterize the lytic transcriptome of HCMV strain Towne varS. We developed a pipeline for transcript annotation using long-read sequencing data. We identified 248 transcriptional start sites, 116 transcriptional termination sites and 80 splicing events. Using this information, we have annotated 291 previously undescribed or only partially annotated transcript isoforms, including eight novel antisense transcripts and their isoforms, as well as a novel transcript (RS2) in the short repeat region, partially antisense to RS1. Similarly to other organisms, we discovered a high transcriptional diversity in HCMV, with many transcripts only slightly differing from one another. Comparing our transcriptome profiling results to an earlier ribosome footprint analysis, we have concluded that the majority of the transcripts contain multiple translationally active ORFs, and also that most isoforms contain unique combinations of ORFs. Based on these results, we propose that one important function of this transcriptional diversity may be to provide a regulatory mechanism at the level of translation.


September 22, 2019

De novo assembly of a Chinese soybean genome.

Soybean was domesticated in China and has become one of the most important oilseed crops. Due to bottlenecks in their introduction and dissemination, soybeans from different geographic areas exhibit extensive genetic diversity. Asia is the largest soybean market; therefore, a high-quality soybean reference genome from this area is critical for soybean research and breeding. Here, we report the de novo assembly and sequence analysis of a Chinese soybean genome for “Zhonghuang 13” by a combination of SMRT, Hi-C and optical mapping data. The assembled genome size is 1.025 Gb with a contig N50 of 3.46 Mb and a scaffold N50 of 51.87 Mb. Comparisons between this genome and the previously reported reference genome (cv. Williams 82) uncovered more than 250,000 structure variations. A total of 52,051 protein coding genes and 36,429 transposable elements were annotated for this genome, and a gene co-expression network including 39,967 genes was also established. This high quality Chinese soybean genome and its sequence analysis will provide valuable information for soybean improvement in the future.


September 22, 2019

KIR3DL01 upregulation on gut natural killer cells in response to SIV infection of KIR- and MHC class I-defined rhesus macaques.

Natural killer cells provide an important early defense against viral pathogens and are regulated in part by interactions between highly polymorphic killer-cell immunoglobulin-like receptors (KIRs) on NK cells and their MHC class I ligands on target cells. We previously identified MHC class I ligands for two rhesus macaque KIRs: KIR3DL01 recognizes Mamu-Bw4 molecules and KIR3DL05 recognizes Mamu-A1*002. To determine how these interactions influence NK cell responses, we infected KIR3DL01+ and KIR3DL05+ macaques with and without defined ligands for these receptors with SIVmac239, and monitored NK cell responses in peripheral blood and lymphoid tissues. NK cell responses in blood were broadly stimulated, as indicated by rapid increases in the CD16+ population during acute infection and sustained increases in the CD16+ and CD16-CD56- populations during chronic infection. Markers of proliferation (Ki-67), activation (CD69 & HLA-DR) and antiviral activity (CD107a & TNFa) were also widely expressed, but began to diverge during chronic infection, as reflected by sustained CD107a and TNFa upregulation by KIR3DL01+, but not by KIR3DL05+ NK cells. Significant increases in the frequency of KIR3DL01+ (but not KIR3DL05+) NK cells were also observed in tissues, particularly in the gut-associated lymphoid tissues, where this receptor was preferentially upregulated on CD56+ and CD16-CD56- subsets. These results reveal broad NK cell activation and dynamic changes in the phenotypic properties of NK cells in response to SIV infection, including the enrichment of KIR3DL01+ NK cells in tissues that support high levels of virus replication.


September 22, 2019

Recent insights into the tick microbiome gained through next-generation sequencing.

The tick microbiome comprises communities of microorganisms, including viruses, bacteria and eukaryotes, and is being elucidated through modern molecular techniques. The advent of next-generation sequencing (NGS) technologies has enabled the genes and genomes within these microbial communities to be explored in a rapid and cost-effective manner. The advantages of using NGS to investigate microbiomes surpass the traditional non-molecular methods that are limited in their sensitivity, and conventional molecular approaches that are limited in their scalability. In recent years the number of studies using NGS to investigate the microbial diversity and composition of ticks has expanded. Here, we provide a review of NGS strategies for tick microbiome studies and discuss the recent findings from tick NGS investigations, including the bacterial diversity and composition, influential factors, and implications of the tick microbiome.


September 22, 2019

Divergent brain gene expression profiles between alternative behavioural helper types in a cooperative breeder.

Juveniles of the cooperatively breeding cichlid fish Neolamprologus pulcher either consistently provide help in form of alloparental egg care (“cleaners”) or consistently abstain from helping (“noncleaners”). These phenotypes are not based on heritable genetic differences. Instead, they arise during ontogeny, which should lead to differences in brain structure or physiology, a currently untested prediction. We compared brain gene expression profiles of cleaners and noncleaners in two experimental conditions, a helping opportunity and a control condition. We aimed to identify (a) expression differences between cleaners and noncleaners in the control, (b) changes in gene expression induced by the opportunity and (c) differences in plasticity of gene expression between cleaners and noncleaners. Control cleaners and noncleaners differed in the expression of a single gene, irx2, which regulates neural differentiation. During the opportunity, cleaners and noncleaners had three upregulated genes in common, which were implicated in neuroplasticity, hormonal signalling and cell proliferation. Thus, the stimulus in the opportunity was sufficiently salient. Cleaners also showed higher expression of seven additional genes that were unique to the opportunity. One of these cleaner-specific genes is implicated in neuropeptide metabolism, indicating that this process is associated with cleaning performance. This suggests that the two types employed different pathways to integrate social information, preparing them for accelerated reaction to future opportunities. Interestingly, three developmental genes were downregulated between the control and the opportunity in cleaners only. Our results indicate that the two behavioural types responded differently to the helping opportunity and that only cleaners responded by downregulating developmental genes.© 2018 John Wiley & Sons Ltd.


September 22, 2019

Identification of a novel fusion transcript between human relaxin-1 (RLN1) and human relaxin-2 (RLN2) in prostate cancer.

Simultaneous expression of highly homologous RLN1 and RLN2 genes in prostate impairs their accurate delineation. We used PacBio SMRT sequencing and RNA-Seq in LNCaP cells in order to dissect the expression of RLN1 and RLN2 variants. We identified a novel fusion transcript comprising the RLN1 and RLN2 genes and found evidence of its expression in the normal and prostate cancer tissues. The RLN1-RLN2 fusion putatively encodes RLN2 isoform with the deleted secretory signal peptide. The identification of the fusion transcript provided information to determine unique RLN1-RLN2 fusion and RLN1 regions. The RLN1-RLN2 fusion was co-expressed with RLN1 in LNCaP cells, but the two gene products were inversely regulated by androgens. We showed that RLN1 is underrepresented in common PCa cell lines in comparison to normal and PCa tissue. The current study brings a highly relevant update to the relaxin field, and will encourage further studies of RLN1 and RLN2 in PCa and broader. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.


September 22, 2019

Improved metagenome assemblies and taxonomic binning using long-read circular consensus sequence data.

DNA assembly is a core methodological step in metagenomic pipelines used to study the structure and function within microbial communities. Here we investigate the utility of Pacific Biosciences long and high accuracy circular consensus sequencing (CCS) reads for metagenomic projects. We compared the application and performance of both PacBio CCS and Illumina HiSeq data with assembly and taxonomic binning algorithms using metagenomic samples representing a complex microbial community. Eight SMRT cells produced approximately 94 Mb of CCS reads from a biogas reactor microbiome sample that averaged 1319 nt in length and 99.7% accuracy. CCS data assembly generated a comparative number of large contigs greater than 1?kb, to those assembled from a ~190x larger HiSeq dataset (~18 Gb) produced from the same sample (i.e approximately 62% of total contigs). Hybrid assemblies using PacBio CCS and HiSeq contigs produced improvements in assembly statistics, including an increase in the average contig length and number of large contigs. The incorporation of CCS data produced significant enhancements in taxonomic binning and genome reconstruction of two dominant phylotypes, which assembled and binned poorly using HiSeq data alone. Collectively these results illustrate the value of PacBio CCS reads in certain metagenomics applications.


September 22, 2019

Unveiling the complexity of the maize transcriptome by single-molecule long-read sequencing.

Zea mays is an important genetic model for elucidating transcriptional networks. Uncertainties about the complete structure of mRNA transcripts limit the progress of research in this system. Here, using single-molecule sequencing technology, we produce 111,151 transcripts from 6 tissues capturing ~70% of the genes annotated in maize RefGen_v3 genome. A large proportion of transcripts (57%) represent novel, sometimes tissue-specific, isoforms of known genes and 3% correspond to novel gene loci. In other cases, the identified transcripts have improved existing gene models. Averaging across all six tissues, 90% of the splice junctions are supported by short reads from matched tissues. In addition, we identified a large number of novel long non-coding RNAs and fusion transcripts and found that DNA methylation plays an important role in generating various isoforms. Our results show that characterization of the maize B73 transcriptome is far from complete, and that maize gene expression is more complex than previously thought.


September 22, 2019

Multiple regulatory networks are activated during cold stress in Medicago sativa L.

Cultivated alfalfa (Medicago sativa L.) is one of the most important perennial legume forages in the world, and it has considerable potential as a valuable forage crop for livestock. However, the molecular mechanisms underlying alfalfa responses to cold stress are largely unknown. In this study, the transcriptome changes in alfalfa under cold stress at 4 °C for 2, 6, 24, and 48 h (three replicates for each time point) were analyzed using the high-throughput sequencing platform, BGISEQ-500, resulting in the identification of 50,809 annotated unigenes and 5283 differentially expressed genes (DEGs). Metabolic pathway enrichment analysis demonstrated that the DEGs were involved in carbohydrate metabolism, photosynthesis, plant hormone signal transduction, and the biosynthesis of amino acids. Moreover, the physiological changes of glutathione and proline content, catalase, and peroxidase activity were in accordance with dynamic transcript profiles of the relevant genes. Additionally, some transcription factors might play important roles in the alfalfa response to cold stress, as determined by the expression pattern of the related genes during 48 h of cold stress treatment. These findings provide valuable information for identifying and characterizing important components in the cold signaling network in alfalfa and enhancing the understanding of the molecular mechanisms underlying alfalfa responses to cold stress.


Talk with an expert

If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.