Dr. Nanda Ghoshal
Principal Scientist, Ramalingaswamy Fellow, Associate Professor (AcSIR)
Assessing the genome sequences of Termitomyces clypeatus for novel metabolite discovery through whole genome sequencing methods and characterization of the metabolites for application in biotechnology
Termitomyces clypeatus belongs to an edible group of mushroom with prized metabolite content [Kumari et al. 2013]. In many parts of Asia, it is used for its medicinal value [Yang et al. 2012]. Termitomyces, belonging to species Basidiomycota, has about 35-40 species, prevalent in tropical parts of Asia and Africa where it has a tight obligatory mutualistic relationship with Termites. Both the Termites and Termitomyces can’t grow apart from each other. Transcriptome sequencing of Termitomyces albuminous [Yang et al 2012] has been carried out revealing about ~6000 transcriptomes. We have sequenced transcriptomes of Termitomyces clypeatus and found about 3 times more number of transcripts than that of T. albuminous [Manuscript under preparation]. Under different treatment conditions the number of transcripts expressed varied by 15% [Out unpublished results]. There are still a large number of metabolic pathway related genes that are not discovered through our transcriptomics studies. In recent years, with rapid advancement in sequencing technologies and falling prices, whole genome sequencing is one of the go to methods for rapid gene and transcript discovery.
In this study we propose to undertake whole genome sequencing of this economically important species using random shutgun methods using Illumina HiSeq platformat about 100X coverage. We will carry out mate pair sequencing with 2 KB and 10 KB insert size for scaffolding purpose. Genome assembly and annotation will be carried out with discovery of novel metabolic pathway genes. We will integrate our transcriptomics data for enriching gene collection and bridging gaps. The production, purification, characterization of the metabolites will be done by cloning and expression of novel glycosyl hydrolases (enzymes) like proteins, of T. clypeatus. The protein activities will be purified and validated by FPLC & HPGPLC and electrophoretic analyses.
Whole Genome and Transcriptome sequencing of selected Indian Cyanobacterial species for candidate Cyanobacterial species for candidate gene discovery for bio-metabolites.
None of the whole genome projects from India grown Cyanobacteria been sequenced and deposited in International Nucleotide Sequence Database collaboration (INSDC) by Indian Institutes to date. India provides 17% of the total biodiversity, and among which the water grown Cyanobacteria are least studied and understood. We have established collaboration with Dept. Of Biotechnology at Visva Bharati that has the largest exotic Cyanobacteria collection in India (Prof. Adhikary – personal communication). We are specifically interested in Cyanobacteria growing under stressed conditions to investigate their gene pool for specific metabolic pathway related genes that can have agricultural significance. We have investigated several pathways in Cyanobacteria in general including that of Nitrogen fixation, lipid metabolism and carbohydrate metabolism [3-5]. We would be interested in discovery of genes from whole genome and transcriptome sequencing data that has agriculturally important properties such as growing under stress conditions and reducing soil alkalinity etc.
Currently we are interested in studying the following six Cyanobacteria species:
- Hassallia byssoidea: Majority of species are aerophytic. Distribution of this species is not very well known due to rampant mis-identifications 
- Mastigocladus laminosus: Grows in hot springs
iii. Scytonema tolypothrichoides: Grows on sub-acrophyte rocks
- Scytonema milli:
- Tolypothrix bouteillei:
- Tolypothrix campylonemoides:
All of these organisms are used as bio fertilizers with the ability to quench alkalinity of the soil. Lack of study in the classification and whole genetic makeup has left big lacunae that we would like to address.
- i) Grow the organisms and sequence the entire genome to study the genetic blue prints of these organisms.
- ii) Use computational methods for predicting genes and constructing metabolic pathways.
iii) Identify gene belonging to novel pathways.
- iv) Create a web portal and host the genomes of the organisms.
- v) Study the transcriptomics and profile them under control and treated conditions.
Developing diagnostic parameters for control of Phytophthora infestans by studying Genetic Diversity using Phenotypic, Moelcular and Nextgen Sequencing methods.
The primary objective is to characterize the P.infestans populations genotypically and phenotypically primarily from West Bengal and secondarily other adjoining states in Eastern India and its correlation so that the information can be used to control future out breaks of late blight in this region. The information would include known hosts, potato and tomato and potential weed hosts if any on which the inoculum is capable of surviving, and other standard phenotypic diversity evaluations. These would include fungicide sensitivity/resistance assays of currently used and newly introduced ones, testing of virulence of the isolates, evaluation of aggressiveness and determination of mating type.
Genotypic diversity analysis would include allozyme diversity at Gpi locus, RG-57 fingerprinting if needed to allow comparisons with lineages studied in the past. However, limited analyses will be performed using RFLPs and allozymes with the major stress being on SSR profiles which will come from use of multiplexed SSR primers. Clustering of SSR profiles will be done with DarWin software and attempt will be made to correlate them to phenotypic traits.
Additionally, the isolates would be screened for sequence diversity of known candidate effectors Avr 3a, Avr–blb2 and the IPI-O family from P. infestans. Due to selection pressure from the hosts, the avirulence genes show extensive variations, including amino acid changes indicative of strong positive selection, gene truncations and deletions, and transcriptional silencing. An attempt would be made to detect this diversity among isolates.
The genotypic and phenotypic assays would be carried out in West Bengal State University, Barasat and all sequencing as well as diversity analysis of the effectors and population genetic analyses would be carried out at Indian Institute of Chemical Biology, Kolkata.
Development of comprehensive genomic resources for newly sequenced genomes
With the reduced cost and rapid pace, the next generation sequencing methods are all set to take over the new data age science. However, there exists a bottleneck since trained human resources and softwares are not developing that fast to transform this data into knowledge. We are also interested in creating novel software resources and piplelines for genome data analysis.
Understanding Genomes of Extremophiles.