About OMICS Group OMICS Group is an amalgamation of Open Access publications and worldwide international science conferences and events.

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1 About OMICS Group OMICS Group is an amalgamation of Open Access publications and worldwide international science conferences and events. Established in the year 2007 with the sole aim of making the information on Sciences and technology Open Access, OMICS Group publishes 500 online open access scholarly journals in all aspects of Science, Engineering, Management and Technology journals. OMICS Group has been instrumental in taking the knowledge on Science & technology to the doorsteps of ordinary men and women. Research Scholars, Students, Libraries, Educational Institutions, Research centers and the industry are main stakeholders that benefitted greatly from this knowledge dissemination. OMICS Group also organizes 500 International conferences annually across the globe, where knowledge transfer takes place through debates, round table discussions, poster presentations, workshops, symposia and exhibitions.

2 About OMICS International Conferences OMICS International is a pioneer and leading science event organizer, which publishes around 500 open access journals and conducts over 500 Medical, Clinical, Engineering, Life Sciences, Pharma scientific conferences all over the globe annually with the support of more than 1000 scientific associations and 30,000 editorial board members and 3.5 million followers to its credit.. OMICS Group has organized 500 conferences, workshops and national symposiums across the major cities including San Francisco, Las Vegas, San Antonio, Omaha, Orlando, Raleigh, Santa Clara, Chicago, Philadelphia, Baltimore, United Kingdom, Valencia, Dubai, Beijing, Hyderabad, Bengaluru and Mumbai.

3 Dr. A. P. J. Abdul Kalam (15 October July 2015)

4 De Novo Transcriptome Assembly and Identification of Cold and Freeze Responsive Genes in Seabuckthorn. Saurabh Chaudhary and Prakash Chand Sharma University School of Biotechnology, Guru Gobind Singh Indraprastha University, Sector -16C, Dwarka, New Delhi

5 Seabuckthorn Hardy, deciduous shrub (family Elaeagnaceae) Yellow or orange berries have tremendous medicinal & nutritional values Indian Himalayas 2 nd richest source in world Prevents soil erosion and helps in land reclamation Grows in extreme environments: important source of new genes (alleles) for abiotic stress tolerance

6 Objectives 1. Seabuckthorn transcriptome assembly and analysis using NGS 2. DeepSAGE based identification of differentially expressed genes during cold and freeze stress 3. Validation of selected cold and freeze responsive genes using qrt-pcr

7 Transcriptome Sequencing 1. Development of seabuckthorn seedlings Seeds were surface sterilized and germinated hydroponically at room temperature After 3 days of germination, seedlings were transferred in growth chamber (28 C, relative humidity 55% and photoperiod 16 hrs light and 8 hrs dark)

8 2. RNA isolation and quality control Total RNA isolated using modified CTAB method with Invisorb Invitek RNA isolation columns Good quality and high yield of total RNA was obtained from leaf and root tissues The RNA samples with A260/A280 ratio from 1.9 to 2.1, A260/A230 ratio from 2.0 to 2.5 and RIN value more than 8.0 were further processed for next generation sequencing

9 3. Transcriptome Assembly High throughput next generation sequencing (Illumina Solexa) platform was used for sequencing transcripts Raw and filtered short read data (NGS QC Tool kit) Item Leaf Root Total number of reads Total number of HQ reads Percentage of HQ reads 91.69% 91.80%

10 Approx. 94 million short reads generated Submitted to NCBI-SRA vide Accession No. SRA Approx. 86 million reads of high quality were further taken for assembly De novo assemblers used to optimize transcriptome assembly Velvet Oases ABySS SOAPdenovo CLC Genomics Workbench Trinity

11 Comparison of de novo assembly as a function of best k-mer Total No. of contigs Average read length N50 read length

12 Comaprision of de novo assembly as a function of additive k-mer Total No. of contigs Average read length N50 read length

13 Comaprision of de novo assembly as a function of additive k-mer followed by TGICL suite Total No. of contigs Average read length N50 read length

14 Comaprision of de novo assembly as a function of ability of short reads to map-back onto seabuckthorn transcriptome Best k-mer Additive k-mer Additive k-mer + TGICL

15 Assembly Parameter Assembly Parameter ABySS Soapdenovo Oases Velvet CLC Trinity Total reads Total bases Min read length Max read length Average read length N50 read length

16 Results On the basis of various assembly parameters, ABySS was found to be the most promising assembly software which resulted into 88,297 putative unigenes in seabuckthorn BLASTX algorithm used to annotate seabuckthorn transcripts (46.8%) had significant hit with existing nr protein database of NCBI. Higher sequence homology was found with Vitis vinifera followed by Ricinus cummunis and Populus trichocarpa. BLAST2GO suite was used to assigned gene ontology terms Functional annotation revealed 3335 unigenes to be involved in different stresses in seabuckthorn transcripts Many transcription factors and simple sequence repeat motifs present in the transcriptome, also identified.

17 Top hit sequence similarity

18 Gene Ontology classification of unigenes (Biological Process)

19 Gene Ontology classification of unigenes (Cellular Component)

20 Gene Ontology classification of unigenes (Molecular Function)

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22 DeepSAGE A combination of classical SAGE (Serial Analysis of Gene Expression) and Next generation Sequencing, used for global gene expression analysis under certain environment Also known as Digital Gene Expression (DGE) Profiling

23 Digital Gene Expression Profiling (DeepSAGE) 30 days old seabuckthorn seedlings were subjected to cold treatment at 4 C and freeze treatment at -10 C for 6 hours Seedlings grown at 26 C were taken as control Leaf tissues were harvested from all three setups Total RNA isolation and quality check from all three samples The 17 base pair tag were generated as per manufacturer protocol for DeepSAGE

24 Digital gene expression (Sequencing and Analysis) Tags were directly sequenced using next generation sequencing (Illumina Solexa) platform Analysis of tags generated revealed large number differentially expressed genes in response to cold and freeze stress. Differentially expressed genes were annotated by sequence similarity search (BLAST) with our existing seabuckthorn transcriptome Around 450 genes identified responsive to cold and freeze stress in seabuckthorn.

25 Pipeline for bioinformatics analysis Raw Sequence Data Non-impurity Data Analysis of Tag Expression and Distribution Analysis of Experimental Reproducibility Clean Tag Data Analysis of Sequencing Saturation Gene Annotation and Normalization Statistics of Gene Expression Analysis of Antisense Transcripts Detection of New Transcript Screening of Differentially Expressed Genes Cluster Analysis of Expression Pattern GO functional Enrichment Analysis Pathway Enrichment Analysis Analysis of Protein- Protein Interaction

26 Distribution of total clean tags (A) & distinct clean tags (B) in DGE sequencing A B

27 Differentially expressed genes (DEGs) Treatment Up regulated genes Down regulated genes Normal vs Cold Stress Normal vs Freeze Stress Cold vs Freeze Stress

28 Gene ontology and KEGG pathway analysis GO- terms suggested metabolic, cellular, primary metabolic, cellular metabolic and macromolecule metabolic processes genes were found to be abundant under cold and freeze stress 28% and 15% of the differentially expressed genes were categorized as genes responsive to stimulus and stress, respectively Photosynthetic, carotenoid biosynthesis and plant hormone signal transduction and vitamin B6 metabolism pathways found to be the most significantly enriched pathways during cold and freeze stress in seabuckthorn.

29 Validation of selected genes (Quantitative real-time RT-PCR) Differentially expressed genes BLAST with existing annotated unigenes of seabuckthorn 22 genes from DeepSAGE data selected (involved in cold and freeze stress for validation) The seabuckthorn seedlings were given various cold and freeze treatments at different time interval Real time expression: biological duplicate and technical triplicates for each samples

30 Expression of 22 genes using qrt-pcr (Validation DeepSAGE) Gene Annotaion Fold change under cold stress MAP2K Mitogen-activated protein kinase kinase ADC Arginine decarboxylase PP2c Protein phosphatase 2c AP2 AP2/ERF domain-containing /ERF transcription factor RPLP0 60s acidic ribosomal protein p ATP19a Peroxidase ATP19a SAUR SAUR family protein HSP70 Heat shock protein WD40 WD-40 repeat family protein KCS Beta-ketoacyl- synthase PAP Plastid-lipid-associated protein Fold change under freeze stress

31 Gene Annotaion Fold change under cold stress RNA-h RNA helicase CALM Calmodulin-related protein SIP2 Galactinol--sucrose galactosyltransferase 6 CSP1 Cold shock protein ABC ATP binding MYO Myosin SIZ1 e3 sumo-protein ligase siz KCS1 3-ketoacyl- synthase I LIP Low temprature induced-like protein HVA 22 HVA 22 E protein SERAT Serine acetyltransferase Fold change under freeze stress

32 Fold Change Expression profiling of genes identified using DeepSAGE (4ºC)

33 Fold Change Expression profiling of genes identified using DeepSAGE (-10ºC)

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35 Conclusions Standardization of high quality RNA isolation from seabuckthorn and establishment of seabuckthorn seedlings in laboratory Whole transcriptome data obtained using next generation sequencing platform provides an upper edge as reference dataset for further studies Unigenes with no relevant hit in NCBI nr protein database were considered to be seabuckthorn specific and can further be characterized in future

36 Standardization of assembly tools for next generation sequencing data helps further in analysis of NGS data for other species Identification of 7421 putative seabuckthorn transcription factor genes Development of USMM for seabuckthorn Many abiotic stress responsive genes, specifically for cold and freeze tolerant in seabuckthorn have been identified.

37 Validation of 20 most important expressed genes during cold and freeze stress, revealed active expression of cold/freeze stress responsible genes in seabuckthorn.

38 Research Leads Genes identified to be involved in cold tolerance need to be further characterized Some of these genes be explored for development of cold tolerant transgenic Assembler (ABySS) optimized for NGS assembly can be used for other similar studies

39 Acknowledgement Department of Biotechnology, Government of India ITS- Department of Science and Technology Government of India Guru Gobind Singh Indraprastha University, India Omics International Conferences

40 Thank You

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