Comparative Genomics Revealed Genus Specific Encoding of Amino Acids by Tri-Nucleotide SSRs in Human Pathogenic Streptococcus and Staphylococus Bacteria

Pathogenic bacteria use phase variation of surface molecules and other characteristics as a significant adaptation mechanism. Repetitive sequences made up of numerous identical repeat units can be found in many phase variable genes. Here, we investigated the frequency and distribution of long-SSRs in 15 human pathogenic Staphylococcus, Streptococcus, and Enterococcus bacteria. Long-SSRs were found to be distributed differently in the genic and intergenic sequences. In the genic sequences, 61.3 SSRs were discovered on average, while 16.2 SSRs were found in the intergenic regions. Staphylococci exhibited the highest frequency of SSRs, followed by Enterococcus, and Streptococci had the lowest frequency of SSRs. Higher A+T content was found to be the best predictor of long-SSR in these human pathogens. Tetranucleotide repeats predominated in intergenic regions, while trinucleotide repeats predominated in genic regions. In human pathogenic Streptococcus and Staphylococcus bacteria, genus-specific encoding of amino acids by tri-nucleotide SSRs was observed. A genetic relationship between these human pathogenic bacteria was derived based on the presence of SSRs in the housekeeping genes and compared to the phylogeny generated based on the 16S ribosomal RNA gene.

Co-circulation of a Novel Dromedary Camel Parainfluenza Virus 3 and Middle East Respiratory Syndrome Coronavirus in a Dromedary Herd With Respiratory Tract Infections

Since the emergence of Middle East Respiratory Syndrome (MERS) in 2012, there have been a surge in the discovery and evolutionary studies of viruses in dromedaries. Here, we investigated a herd of nine dromedary calves from Umm Al Quwain, the United Arab Emirates that developed respiratory signs. Viral culture of the nasal swabs from the nine calves on Vero cells showed two different types of cytopathic effects (CPEs), suggesting the presence of two different viruses. Three samples showed typical CPEs of Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) in Vero cells, which was confirmed by partial RdRp gene sequencing. Complete genome sequencing of the three MERS-CoV strains showed that they belonged to clade B3, most closely related to another dromedary MERS-CoV isolate previously detected in Dubai. They also showed evidence of recombination between lineages B4 and B5 in ORF1ab. Another three samples showed non-typical CPEs of MERS-CoV with cell rounding, progressive degeneration, and detachment. Electron microscopy revealed spherical viral particles with peplomers and diameter of about 170nm. High-throughput sequencing and metagenomic analysis showed that the genome organization (3'-N-P-M-F-HN-L-5') was typical of paramyxovirus. They possessed typical genome features similar to other viruses of the genus Respirovirus, including a conserved motif 323FAPGNYALSYAM336 in the N protein, RNA editing sites 5'-717AAAAAAGGG725-3', and 5'-1038AGAAGAAAGAAAGG1051-3' (mRNA sense) in the P gene with multiple polypeptides coding capacity, a nuclear localization signal sequence 245KVGRMYSVEYCKQKIEK261 in the M protein, a conserved sialic acid binding motif 252NRKSCS257 in the HN protein, conserved lengths of the leader (55nt) and trailer (51nt) sequences, total coding percentages (92.6–93.4%), gene-start (AGGANNAAAG), gene-end (NANNANNAAAAA), and trinucleotide intergenic sequences (CTT, mRNA sense). Phylogenetic analysis of their complete genomes showed that they were most closely related to bovine parainfluenza virus 3 (PIV3) genotype C strains. In the phylogenetic tree constructed using the complete L protein, the branch length between dromedary camel PIV3 (DcPIV3) and the nearest node is 0.04, which is >0.03, the definition used for species demarcation in the family Paramyxoviridae. Therefore, we show that DcPIV3 is a novel species of the genus Respirovirus that co-circulated with MERS-CoV in a dromedary herd in the Middle East.

Long-read sequencing reveals the structural complexity of genomic integration of HBV DNA in hepatocellular carcinoma

The integration of HBV DNA into the human genome can disrupt its structure in hepatocellular carcinoma (HCC), but the complexity of HBV genomic integration remains elusive. Here we applied long-read sequencing to precisely elucidate the HBV integration pattern in the human hepatocellular genome. The DNA library was sequenced using the long-read sequencing on GridION and PacBio Sequel II, respectively. The DNA and mRNA were sequenced using next-generation sequencing on Illumina NextSeq. BLAST (Basic Local Alignment Search Tool) and local scripts were used to analyze HBV integration patterns. We established an analytical strategy based on the long-read sequences, and analyzed the complexity of HBV DNA integration into the hepatocellular genome. A total of 88 integrated breakpoints were identified. HBV DNA integration into human genomic DNA was mainly fragmented with different orientations, rarely with a complete genome. The same HBV integration breakpoints were identified among the three platforms. Most breakpoints were observed at P, X, and S genes in the HBV genome, and observed at introns, intergenic sequences, and exons in the human genome. Tumor tissue harbored a much higher integrated number than the adjacent tissue, and the distribution of HBV integrated into human chromosomes was more concentrated. HBV integration shows different patterns between cancer cells and adjacent normal cells. We for the first time obtained the entire HBV integration pattern through long-read sequencing and demonstrated the value of long-read sequencing in detecting the genomic integration structures of viruses in host cells.

Complete pan-plastome sequences enable high resolution phylogenetic classification of sugar beet and closely related crop wild relatives

As the major source of sugar in moderate climates, sugar-producing beets (Beta vulgaris subsp. vulgaris) have a high economic value. However, the low genetic diversity within cultivated beets requires introduction of new traits, for example to increase their tolerance and resistance attributes - traits that often reside in the crop wild relatives. For this, genetic information of wild beet relatives and their phylogenetic placements to each other are crucial. To answer this need, we sequenced and assembled the complete plastome sequences from a broad species spectrum across the beet genera Beta and Patellifolia, both embedded in the Betoideae (order Caryophyllales). This pan-plastome dataset was then used to determine the wild beet phylogeny in high-resolution. We sequenced the plastomes of 18 closely related accessions representing 11 species of the Betoideae subfamily and provide high-quality plastome assemblies which represent an important resource for further studies of beet wild relatives and the diverse plant order Caryophyllales. Their assembly sizes range from 149,723 bp (Beta vulgaris subsp. vulgaris) to 152,816 bp (Beta nana), with most variability in the intergenic sequences. Combining plastome-derived phylogenies with read-based treatments based on mitochondrial information, we were able to suggest a unified and highly confident phylogenetic placement of the investigated Betoideae species. Our results show that the genus Beta can be divided into the two clearly separated sections Beta and Corollinae. Our analysis confirms the affiliation of B. nana with the other Corollinae species, and we argue against a separate placement in the Nanae section. Within the Patellifolia genus, the two diploid species Patellifolia procumbens and Patellifolia webbiana are, regarding the plastome sequences, genetically more similar to each other than to the tetraploid Patellifolia patellaris. Nevertheless, all three Patellifolia species are clearly separated. In conclusion, our wild beet plastome assemblies represent a new resource to understand the molecular base of the beet germplasm. Despite large differences on the phenotypic level, our pan-plastome dataset is highly conserved. For the first time in beets, our whole plastome sequences overcome the low sequence variation in individual genes and provide the molecular backbone for highly resolved beet phylogenomics. Hence, our plastome sequencing strategy can also guide genomic approaches to unravel other closely related taxa.

Population expansion and genetic structure in Cephalocereus nizandensis (Cactaceae), a microendemic cactus of rocky outcrops of the Tehuantepec basin, Mexico

Background and aims – Cephalocereus nizandensis is a microendemic columnar cactus that grows isolated in xerophytic enclaves associated with rocky outcrops in the Isthmus of Tehuantepec, in the south of Mexico. Its demographic history and genetic structure were assessed to determine the main events that shaped its current restricted distribution.Material and methods – Chloroplast intergenic sequences of 40 individuals and inter simple sequence repeats (ISSRs) of 45 individuals from four isolated populations were used to estimate haplotypic and nucleotide diversity, using expected heterozygosity and the Shannon index. AMOVA, population pair-wise FST, and Bayesian clustering analyses were performed to explore the genetic structure. Demographic history was estimated with neutrality tests, mismatch distribution analysis, and Bayesian skyline plots. Phylogenetic relationships and divergence times were determined using a median joining network and a Bayesian molecular clock.Key results – C. nizandensis has a high diversity and moderate genetic differentiation. The lowest elevation locality was found to be the most genetically distinct. The species has undergone a process of population expansion that began 150,000 years ago and has remained without evidence of a population contraction in the transition from the Pleistocene to the Holocene (11,700 years ago).Conclusions – C. nizandensis presents moderate but significant genetic differentiation, which may be due to an early divergence of its populations. Currently observed levels of genetic diversity are the result of historical maintenance of high population sizes and a population expansion approximately in the last 150,000 years, which was sustained independently of the climatic fluctuations of the Early Quaternary, due in part to the stability of the rocky habitat.

Genetic and Metabolomic Differentiation of Physalis Ixocarpa Brot. Populations in Michoacan State, Mexico

Physalis ixocarpa Brot. is a native species that is consumed in many localities of the Cienega-Chapala in Mexico's Michoacan state. These fruits are cultivated and collected into traditional maize crops. The fruits are similar to P. Philadelphica, but the differences are in the fruit size and organoleptic properties (flavor, sweetness). According to antecedents of domestication that this zone represents in Mexico, is possible that P. ixocarpa shows incipient differentiation signals in genetic structure and metabolomic fingerprinting. Our objective was find evidences of genetic and metabolomic differentiation among populations of P. ixocarpa in the Cienega-Chapala. We used the sequencing of the chloroplast intergenic sequences psbJ – petA and trnL – rpL32, and the metabolomic fingerprinting by GC-MS. The results showed that exist genetic differentiation (FST) and signatures of selection (Fu's Fs' neutrality test) among populations. Moreover the metabolomic fingerprinting showed differences among populations and an increase of aldehydes, aromatic aldehydes, ester, and alcohols related with organoleptic properties of P. ixocarpa. We conclude that P. ixocarpa is an important genetic resource with signatures of differentiation in the Cienega-Chapala, Michoacan state, Mexico that eventually could be related with domestication signatures.

SARS-CoV-2-Host Chimeric RNA-Sequencing Reads Do Not Necessarily Arise From Virus Integration Into the Host DNA

The human genome bears evidence of extensive invasion by retroviruses and other retroelements, as well as by diverse RNA and DNA viruses. High frequency of somatic integration of the RNA virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into the DNA of infected cells was recently suggested, based on a number of observations. One key observation was the presence of chimeric RNA-sequencing (RNA-seq) reads between SARS-CoV-2 RNA and RNA transcribed from human host DNA. Here, we examined the possible origin specifically of human-SARS-CoV-2 chimeric reads in RNA-seq libraries and provide alternative explanations for their origin. Chimeric reads were frequently detected also between SARS-CoV-2 RNA and RNA transcribed from mitochondrial DNA or episomal adenoviral DNA present in transfected cell lines, which was unlikely the result of SARS-CoV-2 integration. Furthermore, chimeric reads between SARS-CoV-2 RNA and RNA transcribed from nuclear DNA were highly enriched for host exonic, rather than intronic or intergenic sequences and often involved the same, highly expressed host genes. Although these findings do not rule out SARS-CoV-2 somatic integration, they nevertheless suggest that human-SARS-CoV-2 chimeric reads found in RNA-seq data may arise during library preparation and do not necessarily signify SARS-CoV-2 reverse transcription, integration in to host DNA and further transcription.

Diverse RNA Viruses Discovered in Three Parasitoid Wasps of the Rice Weevil Sitophilus oryzae

ABSTRACT In this study, many virus-like fragments were obtained from transcriptomes of three wasp species, including Anisopteromalus calandrae (8), Lariophagus distinguendus (3), and Theocolax elegans (18), which can parasitize and control rice weevil Sitophilus oryzae, a serious insect pest of farm-stored grains. By further bioinformatic analysis and sequencing, we identified six novel RNA viruses with complete genomes and named them WWPSRV-1, WWPSRV-2, AcPSRV-1, AcNSRV-1, AcNSRV-2, and LdNSRV-1. PCR-based detection revealed that WWPSRV-1 and WWPSRV-2 had the possibility of interspecies virus transmission, especially WWPSRV-2, which was also present in the rice weevil adults. Phylogenetically, three out of these six viruses appeared to be members of order Picornavirales: WWPSRV-1 belonged to unassigned virus families of this order, whereas WWPSRV-2 and AcPSRV-1 belonged to families Iflaviridae and Dicistroviridae, respectively. The conserved picornavirus-typical domains helicase, protease, and RNA-dependent RNA polymerase could be found in the nonstructural protein encoded by the three viruses, whose genomes consisted of the different numbers of open reading frames (ORFs). The other three RNA viruses could be classified to order Mononegavirales: AcNSRV-1 and AcNSRV-2 belonged to family Lispiviridae, whereas LdNSRV-1 belonged to a big family Rhabdoviridae. The genomes of the three viruses contained at least five ORFs, encoding deduced proteins in the following order: 3′-N-P-M-G-L-5′. All the ORFs were separated by conserved intergenic sequences which likely regulated the transcription termination and initiation. Our findings enhance the understanding of RNA viruses in weevil wasps and set the foundation for the future study of the association among weevils, weevil wasps, and RNA viruses. IMPORTANCE The enormous diversity of RNA viruses in insects is continuously validated. Parasitoid wasps, as biocontrol insects which are widely used against insect pests in agroecosystems, may also carry many “good” RNA viruses. Some RNA viruses in parasitoid wasps have been reported to affect the host wasps or the wasps’ host. Here, six novel RNA viruses with complete genomes were identified in three parasitoid wasps of the rice weevil. One of these viruses was also detected in the rice weevil adults. Phylogenetically, WWPSRV-1 was the first unambiguous detection of Nora-like virus in insect parasitoids. WWPSRV-2 and AcPSRV-1 belong to families Iflaviridae and Dicistroviridae, some viruses of which can result in lethal infections in silkworms and honeybees. The other three RNA viruses belong to order Mononegavirales, which comprises many well-known insect-associated viruses.

BIOLOGICAL GENE SEQUENCE STUCTURE ANALYSIS USING HIDDEN MARKOV MODEL

Identification or prediction of coding sequences from within genomic DNA has been a major part of the search of the gene. In this work real hidden Markov models (HMMs) to denote the consensus and deliver a beneficial tool in determining the splicing junction sites Markov models which has a recurring nature in computational biology leads to statistical models, in every sequential analysis it plays a role of putting up a right label on each residue. In sequential alignment and as well as in gene identification namely exons, introns or intergenic sequences which make in a sequence with homologous residue with the target database. Under the gene identification methodology Condon bias, exons, introns have length preference which leads to a combination of splice site consensus. Parameters are fixed on the onset while weight of the different information are polled together leading to the interception of result probability, which could lead to identifying the best score based on score mean and how confident are the best scoring answers are perfect. This leads to the concept of extendibility, to perfect and ad hoc gene finder, which is a modeled transitional methodology leading to the consensus, alternate splicing and offers polyadenylation signal. This leads to piling of authenticity against a delicate ad hoc program which could make to breakdown under its individual weightiness.

SARS-CoV-2-host chimeric RNA-sequencing reads do not necessarily signify virus integration into the host DNA

ABSTRACTThe human genome bears evidence of extensive invasion by retroviruses and other retroelements, as well as by diverse RNA and DNA viruses. High frequency of somatic integration of the RNA virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into the DNA of infected cells was recently suggested, partly based on the detection of chimeric RNA-sequencing (RNA-seq) reads between SARS-CoV-2 RNA and RNA transcribed from human host DNA. Here, we examined the possible origin of human-SARS-CoV-2 chimeric reads in RNA-seq libraries and provide alternative explanations for their origin. Chimeric reads were frequently detected also between SARS-CoV-2 RNA and RNA transcribed from mitochondrial DNA or episomal adenoviral DNA present in transfected cell lines, which was unlikely the result of SARS-CoV-2 integration. Furthermore, chimeric reads between SARS-CoV-2 RNA and RNA transcribed from nuclear DNA was highly enriched for host exonic, than intronic or intergenic sequences and often involved the same, highly expressed host genes. These findings suggest that human-SARS-CoV-2 chimeric reads found in RNA-seq data may arise during library preparation and do not necessarily signify SARS-CoV-2 reverse transcription, integration in to host DNA and further transcription.