De novo genome assembly and annotation of the medicinal plant Tinospora cordifolia (Willd.) Miers ex Hook. f. & Thom's.

Indian natural climbing shrub Tinospora cordifolia, often known as "Guduchi" and "Amrita," is a highly esteemed medicinal plant in the Indian system of medicine (ISM). It is a member of the Menispermaceae family which consists of a rich source of protein, micronutrients, and rich source of bioactive components which are used in treating various systemic diseases. The current study was designed to know the biological characterization of the plant genome and biosynthesis of plant metabolites essential for its medicinal applications. Tinospora cordifolia's complete genome was sequenced using Illumina HiSeq2500 sequencing technology. The draft genome was assembled through a de novo method. An integrative genome annotation approach was used to perform functional gene prediction. The pathway analysis was carried out using the KEGG database. The total genome size obtained after genome assembly was 894 Mb with an N50 of 9148 bp. The integrative annotation approach resulted in 35,111 protein-coding genes. In addition, genes responsible for the synthesis of syringin, a secondary metabolite found in plants, were identified. In comparison to the standard drug (dopamine, rasagiline, and selegiline), syringin's molecular docking exhibited a greater binding affinity from the range of - 4.3 to - 6.6 kcal/mol for all the targets of Parkinson's disease and for Alzheimer's targets; it has shown the maximum potency from the range of - 6.5 to - 7.4 kcal/mol with respect to the standard drug (donepezil, galantamine, and rivastigmine). This study provides the genomic information of Tinospora cordifolia which is helpful in understanding genomic insights and metabolic pathways connected to the corresponding plant genome and predicts the possible useful effect for the molecular characterization of therapeutic drugs.

Genomic and transcriptomic analysis of sacred fig (Ficus religiosa).

BACKGROUND: Peepal/Bodhi tree (Ficus religiosa L.) is an important, long-lived keystone ecological species. Communities on the Indian subcontinent have extensively employed the plant in Ayurveda, traditional medicine, and spiritual practices. The Peepal tree is often thought to produce oxygen both during the day and at night by Indian folks. The goal of our research was to produce molecular resources using whole-genome and transcriptome sequencing techniques. RESULTS: The complete genome of the Peepal tree was sequenced using two next-generation sequencers Illumina HiSeq1000 and MGISEQ-2000. We assembled the draft genome of 406 Mb, using a hybrid assembly workflow. The genome annotation resulted in 35,093 protein-coding genes; 53% of its genome consists of repetitive sequences. To understand the physiological pathways in leaf tissues, we analyzed photosynthetically distinct conditions: bright sunny days and nights. The RNA-seq analysis supported the expression of 26,479 unigenes. The leaf transcriptomic analysis of the diurnal and nocturnal periods revealed the expression of the significant number of genes involved in the carbon-fixation pathway. CONCLUSIONS: This study presents a draft hybrid genome assembly for F. religiosa and its functional annotated genes. The genomic and transcriptomic data-derived pathways have been analyzed for future studies on the Peepal tree.

De novo genome assembly and annotation of gall-forming medicinal plant Pistacia chinensis subsp. integerrima (J. L. Stewart ex Brandis) Rech. f.

Pistacia chinensis subsp. integerrima is one of the medicinal plants, well known for gall formation and popularly used in Ayurveda to treat various systemic diseases such as chronic disorders, respiratory problems, etc. P. integerrima genome characterization will aid in the study of Pistacia genes and pathways involved in therapeutic application. To understand the biological characteristics of this plant and to gain the genetic insight into the biosynthesis of its natural compounds, the whole genome of P. integerrima and its leaf transcriptome was sequenced using Illumina sequencing technology. The sequenced genome was functionally annotated, and gene prediction was performed with integrated genome annotation workflow. The pathway analysis was carried out using KEGG database. We obtained a draft genome assembly of 462 Mb with N50 16,145 bp. A total of 39,452 genes were found, and 18,492 of these contained RNA or protein evidence. We characterized the genes involved in biosynthetic pathways of different plant secondary metabolites such as flavonoids and terpenoids. Also, we identified miR397 and miR828 family noncoding RNA; which mainly targets the laccase (LCA) and MYB protein functioning respectively. Phylogeneic analysis showed that P. integerrima is genetically more closer to P. vera. In this study, we attempt to explore the whole genome information of P. integerrima which will provide a genomic insight in the future for omics studies as well as serves as valuable resource for the molecular characterization of medicinal compounds.

In planta transcriptome analysis reveals tissue-specific expression of pathogenicity genes and microRNAs during rice-Magnaporthe interactions.

Transcriptional re-programming in host and pathogen upon leaf and neck infection is an evolving area of research for the rice blast community. Analysis of in planta rice transcriptome in leaf and neck tissues revealed tissue-specific and infection-specific expression of rice and Magnaporthe oryzae genes in host and pathogen. The glycosyl hydrolase, isocitrate lyase, cupin domain containing protein, TF2, CMPG1, CHIT17 and OsCML14 genes were uniquely expressed in leaf infection. Genes like cytochrome P450, inhibitor I family protein, GSTU6, abscisic stress ripening, and cupin domain containing protein were up-regulated during neck infection. In our microRNA sequencing study, Osa-miR166n-3p was highly expressed in upon Magnaporthe leaf infection, whereas osa-miR1661-3p, osa-miR166n-3p and osa-miR159b were overexpressed in neck infection. Here we report several transcripts being targeted by up and down regulated microRNAs during infection. The putative genes expressed upon infection in leaf and neck could be used in understanding the dual-epidemics of blast disease.

Comparative analysis of secondary metabolite gene clusters in different strains of Magnaporthe oryzae.

Rice blast caused by Magnaporthe oryzae continues to be a major constraint in rice production worldwide. Rice is one of the staple crops in India and rice blast causes huge economic losses. Interestingly, the Indian subcontinent is the centre for origin and diversity of rice as well as the Magnaporthe species complex. Secondary metabolites are known to play important role in pathogenesis and M. oryzae has high potential of genes involved in secondary metabolism but, unfortunately most of them remain uncharacterized. In the present study, we analysed the draft genome assemblies of M. oryzae strains isolated from different parts of India, for putative secondary metabolite key gene (SMKG) clusters encoding polyketide synthases, non-ribosomal peptide synthetases, diterpene cyclases and dimethylallyl tryptophan synthase. Based on the complete genome sequence of 70-15 strain and its previous reports of identified SMKGs, we have identified the key genes for the interrogated strains. Expression analysis of these genes amongst different strains indicates how they have evolved depending on the host and environmental conditions. To our knowledge, this study is first of its kind where the secondary metabolism genes and their role in functional adaptation were studied across several strains of M. oryzae.

High-Resolution Full-Length HLA Typing Method Using Third Generation (Pac-Bio SMRT) Sequencing Technology.

The human HLA genes are among the most polymorphic genes in the human genome. Therefore, it is very difficult to find two unrelated individuals with identical HLA molecules. As a result, HLA Class I and Class II genes are routinely sequenced or serotyped for organ transplantation, autoimmune disease-association studies, drug hypersensitivity research, and other applications. However, these methods were able to give two or four digit data, which was not sufficient enough to understand the completeness of haplotypes of HLA genes. To overcome these limitations, we here described end-to-end workflow for sequencing of HLA class I and class II genes using third generation sequencing, SMRT technology. This method produces fully-phased, unambiguous, allele-level information on the PacBio System.

Recovery of Five Complete Influenza A(H1N1)pdm09 Genome Sequences from the 2015 Influenza Outbreak in India by Metagenomic Sequencing.

Five complete (H1N1)pdm09 viral sequences were recovered from hospitalized individuals during the 2015 influenza outbreak by metagenomic sequencing. Four of the genomes are from oropharyngeal swabs, and one is from an isolate. All five sequences belong to an emerging 6B clade. Studying them further is critical for outbreak preparedness.

Multi-Omics Driven Assembly and Annotation of the Sandalwood (Santalum album) Genome.

Indian sandalwood (Santalum album) is an important tropical evergreen tree known for its fragrant heartwood-derived essential oil and its valuable carving wood. Here, we applied an integrated genomic, transcriptomic, and proteomic approach to assemble and annotate the Indian sandalwood genome. Our genome sequencing resulted in the establishment of a draft map of the smallest genome for any woody tree species to date (221 Mb). The genome annotation predicted 38,119 protein-coding genes and 27.42% repetitive DNA elements. In-depth proteome analysis revealed the identities of 72,325 unique peptides, which confirmed 10,076 of the predicted genes. The addition of transcriptomic and proteogenomic approaches resulted in the identification of 53 novel proteins and 34 gene-correction events that were missed by genomic approaches. Proteogenomic analysis also helped in reassigning 1,348 potential noncoding RNAs as bona fide protein-coding messenger RNAs. Gene expression patterns at the RNA and protein levels indicated that peptide sequencing was useful in capturing proteins encoded by nuclear and organellar genomes alike. Mass spectrometry-based proteomic evidence provided an unbiased approach toward the identification of proteins encoded by organellar genomes. Such proteins are often missed in transcriptome data sets due to the enrichment of only messenger RNAs that contain poly(A) tails. Overall, the use of integrated omic approaches enhanced the quality of the assembly and annotation of this nonmodel plant genome. The availability of genomic, transcriptomic, and proteomic data will enhance genomics-assisted breeding, germplasm characterization, and conservation of sandalwood trees.

Complete assembly of a dengue virus type 3 genome from a recent genotype III clade by metagenomic sequencing of serum.

Background: Mosquito-borne flaviviruses, such as dengue and Japanese encephalitis virus (JEV), cause life-threatening diseases, particularly in the tropics. Methods: Here we performed unbiased metagenomic sequencing of RNA extracted from the serum of four patients and the plasma of one patient, all hospitalized at a tertiary care centre in South India with severe or prolonged febrile illness, together with the serum from one healthy control, in 2014. Results: We identified and assembled a complete dengue virus type 3 sequence from a case of severe dengue fever. We also identified a small number of JEV sequences in the serum of two adults with febrile illness, including one with severe dengue. Phylogenetic analysis revealed that the dengue sequence belonged to genotype III. It has an estimated divergence time of 13.86 years from the most highly related Indian strains. In total, 11 amino acid substitutions were predicted for this strain in the antigenic envelope protein, when compared to the parent strain used for development of the first commercial dengue vaccine.  Conclusions: We demonstrate that both genome assembly and detection of a low number of viral sequences are possible through the unbiased sequencing of clinical material. These methods may help ascertain causal agents for febrile illnesses with no known cause.

Comparative Metagenomics Reveal Phylum Level Temporal and Spatial Changes in Mycobiome of Belowground Parts of Crocus sativus.

Plant-fungal associations have been explored by routine cultivation based approaches and cultivation based approaches cannot catalogue more than 5% of fungal diversity associated with any niche. In the present study, an attempt has been made to catalogue fungal diversity associated with belowground parts i.e. rhizosphere and cormosphere, of Crocus sativus (an economically important herb) during two growth stages, using cultivation independent ITS gene targeted approach, taking bulk soil as reference. The 454 pyrosequencing sequence data analysis suggests that the fungal diversity was niche and growth stage specific. Fungi diversity, in the present case, was not only different between the two organs (roots and corm) but the dominance pattern varies between the cormosphere during two growth stages. Zygomycota was dominant fungal phylum in the rhizosphere whereas Basidiomycota was dominant in cormosphere during flowering stage. However in cormosphere though Basidiomycota was dominant phylum during flowering stage but Zygomycota was dominant during dormant stage. Interestingly, in cormosphere, the phyla which was dominant at dormant stage was rare at flowering stage and vice-versa (Basidiomycota: Flowering = 93.2% Dormant = 0.05% and Zygomycota: Flowering = 0.8% Dormant = 99.7%). At genus level, Rhizopus was dominant in dormant stage but was rare in flowering stage (Rhizopus: Dormant = 99.7% Flowering = 0.55%). This dynamics is not followed by the bulk soil fungi which was dominated by Ascomycota during both stages under study. The genus Fusarium, whose species F. oxysporum causes corm rot in C. sativus, was present during both stages with slightly higher abundance in roots. Interestingly, the abundance of Rhizopus varied a great deal in two stages in cormosphere but the abundance of Fusarium was comparable in two growth stages (Bulk soil Flowering = 0.05%, Rhizosphere Flowering = 1.4%, Cormosphere Flowering = 0.06%, Bulk soil Dormant = 2.47% and cormosphere dormant = 0.05%). This is the first report on the fungal diversity associated with the root of Crocus sativus and first report on the fungi associated with corm of any plant with the temporal and spatial variation in the fungal community structure.

Genome-Wide Comparison of Magnaporthe Species Reveals a Host-Specific Pattern of Secretory Proteins and Transposable Elements.

Blast disease caused by the Magnaporthe species is a major factor affecting the productivity of rice, wheat and millets. This study was aimed at generating genomic information for rice and non-rice Magnaporthe isolates to understand the extent of genetic variation. We have sequenced the whole genome of the Magnaporthe isolates, infecting rice (leaf and neck), finger millet (leaf and neck), foxtail millet (leaf) and buffel grass (leaf). Rice and finger millet isolates infecting both leaf and neck tissues were sequenced, since the damage and yield loss caused due to neck blast is much higher as compared to leaf blast. The genome-wide comparison was carried out to study the variability in gene content, candidate effectors, repeat element distribution, genes involved in carbohydrate metabolism and SNPs. The analysis of repeat element footprints revealed some genes such as naringenin, 2-oxoglutarate 3-dioxygenase being targeted by Pot2 and Occan, in isolates from different host species. Some repeat insertions were host-specific while other insertions were randomly shared between isolates. The distributions of repeat elements, secretory proteins, CAZymes and SNPs showed significant variation across host-specific lineages of Magnaporthe indicating an independent genome evolution orchestrated by multiple genomic factors.

Indica rice genome assembly, annotation and mining of blast disease resistance genes.

BACKGROUND: Rice is a major staple food crop in the world. Over 80% of rice cultivation area is under indica rice. Currently, genomic resources are lacking for indica as compared to japonica rice. In this study, we generated deep-sequencing data (Illumina and Pacific Biosciences sequencing) for one of the indica rice cultivars, HR-12 from India. RESULTS: We assembled over 86% (389 Mb) of rice genome and annotated 56,284 protein-coding genes from HR-12 genome using Illumina and PacBio sequencing. Comprehensive comparative analyses between indica and japonica subspecies genomes revealed a large number of indica specific variants including SSRs, SNPs and InDels. To mine disease resistance genes, we sequenced few indica rice cultivars that are reported to be highly resistant (Tetep and Tadukan) and susceptible (HR-12 and Co-39) against blast fungal isolates in many countries including India. Whole genome sequencing of rice genotypes revealed high rate of mutations in defense related genes (NB-ARC, LRR and PK domains) in resistant cultivars as compared to susceptible. This study has identified R-genes Pi-ta and Pi54 from durable indica resistant cultivars; Tetep and Tadukan, which can be used in marker assisted selection in rice breeding program. CONCLUSIONS: This is the first report of whole genome sequencing approach to characterize Indian rice germplasm. The genomic resources from our work will have a greater impact in understanding global rice diversity, genetics and molecular breeding.

Genome analysis of rice-blast fungus Magnaporthe oryzae field isolates from southern India.

The Indian subcontinent is the center of origin and diversity for rice (Oryza sativa L.). The O. sativa ssp. indica is a major food crop grown in India, which occupies the first and second position in area and production, respectively. Blast disease caused by Magnaporthe oryzae is a major constraint to rice production. Here, we report the analysis of genome architecture and sequence variation of two field isolates, B157 and MG01, of the blast fungus from southern India. The 40 Mb genome of B157 and 43 Mb genome of MG01 contained 11,344 and 11,733 predicted genes, respectively. Genomic comparisons unveiled a large set of SNPs and several isolate specific genes in the Indian blast isolates. Avr genes were analyzed in several sequenced Magnaporthe strains; this analysis revealed the presence of Avr-Pizt and Avr-Ace1 genes in all the sequenced isolates. Availability of whole genomes of field isolates from India will contribute to global efforts to understand genetic diversity of M. oryzae population and to track the emergence of virulent pathotypes.

Comprehensive analyses of genomes, transcriptomes and metabolites of neem tree.

Neem (Azadirachta indica A. Juss) is one of the most versatile tropical evergreen tree species known in India since the Vedic period (1500 BC-600 BC). Neem tree is a rich source of limonoids, having a wide spectrum of activity against insect pests and microbial pathogens. Complex tetranortriterpenoids such as azadirachtin, salanin and nimbin are the major active principles isolated from neem seed. Absolutely nothing is known about the biochemical pathways of these metabolites in neem tree. To identify genes and pathways in neem, we sequenced neem genomes and transcriptomes using next generation sequencing technologies. Assembly of Illumina and 454 sequencing reads resulted in 267 Mb, which accounts for 70% of estimated size of neem genome. We predicted 44,495 genes in the neem genome, of which 32,278 genes were expressed in neem tissues. Neem genome consists about 32.5% (87 Mb) of repetitive DNA elements. Neem tree is phylogenetically related to citrus, Citrus sinensis. Comparative analysis anchored 62% (161 Mb) of assembled neem genomic contigs onto citrus chromomes. Ultrahigh performance liquid chromatography-mass spectrometry-selected reaction monitoring (UHPLC-MS/SRM) method was used to quantify azadirachtin, nimbin, and salanin from neem tissues. Weighted Correlation Network Analysis (WCGNA) of expressed genes and metabolites resulted in identification of possible candidate genes involved in azadirachtin biosynthesis pathway. This study provides genomic, transcriptomic and quantity of top three neem metabolites resource, which will accelerate basic research in neem to understand biochemical pathways.

Genome sequencing of herb Tulsi (Ocimum tenuiflorum) unravels key genes behind its strong medicinal properties.

BACKGROUND: Krishna Tulsi, a member of Lamiaceae family, is a herb well known for its spiritual, religious and medicinal importance in India. The common name of this plant is 'Tulsi' (or 'Tulasi' or 'Thulasi') and is considered sacred by Hindus. We present the draft genome of Ocimum tenuiflurum L (subtype Krishna Tulsi) in this report. The paired-end and mate-pair sequence libraries were generated for the whole genome sequenced with the Illumina Hiseq 1000, resulting in an assembled genome of 374 Mb, with a genome coverage of 61 % (612 Mb estimated genome size). We have also studied transcriptomes (RNA-Seq) of two subtypes of O. tenuiflorum, Krishna and Rama Tulsi and report the relative expression of genes in both the varieties. RESULTS: The pathways leading to the production of medicinally-important specialized metabolites have been studied in detail, in relation to similar pathways in Arabidopsis thaliana and other plants. Expression levels of anthocyanin biosynthesis-related genes in leaf samples of Krishna Tulsi were observed to be relatively high, explaining the purple colouration of Krishna Tulsi leaves. The expression of six important genes identified from genome data were validated by performing q-RT-PCR in different tissues of five different species, which shows the high extent of urosolic acid-producing genes in young leaves of the Rama subtype. In addition, the presence of eugenol and ursolic acid, implied as potential drugs in the cure of many diseases including cancer was confirmed using mass spectrometry. CONCLUSIONS: The availability of the whole genome of O.tenuiflorum and our sequence analysis suggests that small amino acid changes at the functional sites of genes involved in metabolite synthesis pathways confer special medicinal properties to this herb.

Identification and characterization of in planta-expressed secreted effector proteins from Magnaporthe oryzae that induce cell death in rice.

Interactions between rice and Magnaporthe oryzae involve the recognition of cellular components and the exchange of complex molecular signals from both partners. How these interactions occur in rice cells is still elusive. We employed robust-long serial analysis of gene expression, massively parallel signature sequencing, and sequencing by synthesis to examine transcriptome profiles of infected rice leaves. A total of 6,413 in planta-expressed fungal genes, including 851 genes encoding predicted effector proteins, were identified. We used a protoplast transient expression system to assess 42 of the predicted effector proteins for the ability to induce plant cell death. Ectopic expression assays identified five novel effectors that induced host cell death only when they contained the signal peptide for secretion to the extracellular space. Four of them induced cell death in Nicotiana benthamiana. Although the five effectors are highly diverse in their sequences, the physiological basis of cell death induced by each was similar. This study demonstrates that our integrative genomic approach is effective for the identification of in planta-expressed cell death-inducing effectors from M. oryzae that may play an important role facilitating colonization and fungal growth during infection.

Draft genome sequence of Staphylococcus aureus ST672, an emerging disease clone from India.

We report the draft genome sequence of methicillin-resistant Staphylococcus aureus (MRSA) strain ST672, an emerging disease clone in India, from a septicemia patient. The genome size is about 2.82 Mb with 2,485 open reading frames (ORFs). The staphylococcal cassette chromosome mec (SCCmec) element (type V) and immune evasion cluster appear to be different from those of strain ST772 on preliminary examination.

Draft genome sequence of Staphylococcus aureus 118 (ST772), a major disease clone from India.

We report the draft genome sequence of an ST772 Staphylococcus aureus disease isolate carrying staphylococcal cassette chromosome mec (SCCmec) type V from a pyomyositis patient. Our de novo short read assembly is ∼2.8 Mb and encodes a unique Panton-Valentine leukocidin (PVL) phage with structural genes similar to those of ϕ7247PVL and novel lysogenic genes at the N termini.

Comparative transcriptomics of three Poaceae species reveals patterns of gene expression evolution.

The Poaceae family, also known as the grasses, includes agronomically important cereal crops such as rice, maize, sorghum, and wheat. Previous comparative studies have shown that much of the gene content is shared among the grasses; however, functional conservation of orthologous genes has yet to be explored. To gain an understanding of the genome-wide patterns of evolution of gene expression across reproductive tissues, we employed a sequence-based approach to compare analogous transcriptomes in species representing three Poaceae subgroups including the Pooideae (Brachypodium distachyon), the Panicoideae (sorghum), and the Ehrhartoideae (rice). Our transcriptome analyses reveal that only a fraction of orthologous genes exhibit conserved expression patterns. A high proportion of conserved orthologs include genes that are upregulated in physiologically similar tissues such as leaves, anther, pistil, and embryo, while orthologs that are highly expressed in seeds show the most diverged expression patterns. More generally, we show that evolution of gene expression profiles and coding sequences in the grasses may be linked. Genes that are highly and broadly expressed tend to be conserved at the coding sequence level while genes with narrow expression patterns show accelerated rates of sequence evolution. We further show that orthologs in syntenic genomic blocks are more likely to share correlated expression patterns compared with non-syntenic orthologs. These findings are important for agricultural improvement because sequence information is transferred from model species, such as Brachypodium, rice, and sorghum to crop plants without sequenced genomes.

Diverse and tissue-enriched small RNAs in the plant pathogenic fungus, Magnaporthe oryzae.

BACKGROUND: Emerging knowledge of the impact of small RNAs as important cellular regulators has prompted an explosion of small transcriptome sequencing projects. Although significant progress has been made towards small RNA discovery and biogenesis in higher eukaryotes and other model organisms, knowledge in simple eukaryotes such as filamentous fungi remains limited. RESULTS: Here, we used 454 pyrosequencing to present a detailed analysis of the small RNA transcriptome (~ 15 - 40 nucleotides in length) from mycelia and appressoria tissues of the rice blast fungal pathogen, Magnaporthe oryzae. Small RNAs mapped to numerous nuclear and mitochondrial genomic features including repetitive elements, tRNA loci, rRNAs, protein coding genes, snRNAs and intergenic regions. For most elements, small RNAs mapped primarily to the sense strand with the exception of repetitive elements to which small RNAs mapped in the sense and antisense orientation in near equal proportions. Inspection of the small RNAs revealed a preference for U and suppression of C at position 1, particularly for antisense mapping small RNAs. In the mycelia library, small RNAs of the size 18 - 23 nt were enriched for intergenic regions and repetitive elements. Small RNAs mapping to LTR retrotransposons were classified as LTR retrotransposon-siRNAs (LTR-siRNAs). Conversely, the appressoria library had a greater proportion of 28 - 35 nt small RNAs mapping to tRNA loci, and were classified as tRNA-derived RNA fragments (tRFs). LTR-siRNAs and tRFs were independently validated by 3' RACE PCR and northern blots, respectively. CONCLUSIONS: Our findings suggest M. oryzae small RNAs differentially accumulate in vegetative and specialized-infection tissues and may play an active role in genome integrity and regulating growth and development.