APPLICATIONS OF SEQUENCE INFORMATION

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APPLICATIONS OF SEQUENCE INFORMATION IN PLANT GENOME ANALYSIS Classical And Advanced Approaches APPLICATIONS OF SEQUENCE INFORMATION IN PLANT GENOME ANALYSIS Classical And Advanced Approaches Geetika 2012A41D

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DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule. It includes any method or technology that is used to determine the order of the four bases—adenine, guanine, cytosine, and thymine—in a strand of DNA. APPLICATIONS OF SEQUENCE INFORMATION IN PLANT GENOME ANALYSIS Classical And Advanced Approaches DNA Sequencing

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APPLICATIONS OF SEQUENCE INFORMATION IN PLANT GENOME ANALYSIS Classical And Advanced Approaches DNA Sequencing Knowledge of DNA sequences has become indispensable for basic biological research, and in numerous applied fields such as : diagnostic biotechnology forensic biology biological systematics

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APPLICATIONS OF SEQUENCE INFORMATION IN PLANT GENOME ANALYSIS Classical And Advanced Approaches Genomics is the study of the genomes of organisms. The field includes intensive efforts to determine the entire DNA sequence of organisms and fine-scale genetic mapping efforts. Genomics was established by Fred Sanger when he first sequenced the complete genomes of a virus and a mitochondrion. His group established techniques of sequencing, genome mapping, data storage, and bioinformatics analyses in the 1970-1980s. Genomics

Applications of DNA Sequencing:

APPLICATIONS OF SEQUENCE INFORMATION IN PLANT GENOME ANALYSIS Classical And Advanced Approaches Applications of DNA Sequencing Forensic Diagnostics Medicine Agriculture Fingerprinting Patenting Registration of a genotype SNP detection Primer designing Mutant Detection QTL analysis Biotechnology Genome sequencing Genome data cards

Applications of DNA Sequencing:

APPLICATIONS OF SEQUENCE INFORMATION IN PLANT GENOME ANALYSIS Classical And Advanced Approaches Applications of DNA Sequencing Forensic Identification of a particular individual/criminal based upon DNA fingerprinting profile of a hair, nail, skin, or blood sample obtained from the site of crime. Solving paternity cases Identification of endangered or protected species

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Forensic Diagnostics Medicine Agriculture Fingerprinting Patenting Registration of a genotype SNP detection Primer designing Mutant Detection QTL analysis Biotechnology Genome sequencing Genome data cards DNA profile of released varieties DNA profile of elite Germplasm lines Required for registration of genotype May be used for claiming patenting rights on the genotype

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Forensic Diagnostics Medicine Agriculture Fingerprinting Patenting Registration of a genotype SNP detection Primer designing Mutant Detection QTL analysis Biotechnology Genome sequencing Genome data cards Sequence information is required for patenting Genes sequences are must for filing any intellectual copyright

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Forensic Diagnostics Medicine Agriculture Fingerprinting Patenting Registration of a genotype SNP detection Primer designing Mutant Detection QTL analysis Biotechnology Genome sequencing Genome data cards Same concept as that of patenting

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Forensic Diagnostics Medicine Agriculture Fingerprinting Patenting Registration of a genotype SNP detection Primer designing Mutant Detection QTL analysis Biotechnology Genome sequencing Genome data cards Gene sequences are compared on the BLAST to detect SNPs Comparative genomic studies To study phylogeny (evolutionary paths)

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Forensic Diagnostics Medicine Agriculture Fingerprinting Patenting Registration of a genotype SNP detection Primer designing Mutant Detection QTL analysis Biotechnology Genome sequencing Genome data cards SSR-Flanking sequence information is required to design primers Random primers by various private companies are designed and sold Gene specific primers are designed

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Forensic Diagnostics Medicine Agriculture Fingerprinting Patenting Registration of a genotype SNP detection Primer designing Mutant Detection QTL analysis Biotechnology Genome sequencing Genome data cards Micro mutants can be identified by comparing gene sequences of mutant and the wild type. Mutations can also be induced using targeted mutagenesis and later sequencing of mutants may indicate type of mutation induced.

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Forensic Diagnostics Medicine Agriculture Fingerprinting Patenting Registration of a genotype SNP detection Primer designing Mutant Detection QTL analysis Biotechnology Genome sequencing Genome data cards QTL mapping is done using recombinant and segregating populations. Mapped QTLs can be sequenced and may be used to create transgenics through MAS.

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Forensic Diagnostics Medicine Agriculture Fingerprinting Patenting Registration of a genotype SNP detection Primer designing Mutant Detection QTL analysis Biotechnology Genome sequencing Genome data cards Creation of transgenics Gene tagging Marker-assisted breeding Fishing out useful genes using FISH technique and confirmation of introgressed genes eg ., rye genome segments in wheat genome

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Forensic Diagnostics Medicine Agriculture Fingerprinting Patenting Registration of a genotype SNP detection Primer designing Mutant Detection QTL analysis Biotechnology Genome sequencing Genome data cards Arabidopsis Rice Sorghum Human

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Forensic Diagnostics Medicine Agriculture Fingerprinting Patenting Registration of a genotype SNP detection Primer designing Mutant Detection QTL analysis Biotechnology Genome sequencing Genome data cards Will contain genome sequence Pedigree records Disease diagnostics Medical treatment All personal data record Genome ID

Applications of DNA Sequencing:

APPLICATIONS OF SEQUENCE INFORMATION IN PLANT GENOME ANALYSIS Classical And Advanced Approaches Applications of DNA Sequencing Medical Science and Diagnostics A Personal Genome ID Card includes a card member and a machine-readable storage medium integrated in the card member based on DNA information of the person. It includes machine-readable data, which represents a sequence of nucleotide bases for at least a portion of a genome of an individual already stored on the machine-readable storage medium. This machine-readable data also include medical history of the individual, and his genetic pedigree information.

Applications of DNA Sequencing:

APPLICATIONS OF SEQUENCE INFORMATION IN PLANT GENOME ANALYSIS Classical And Advanced Approaches Applications of DNA Sequencing A Plant Variety ID Card Will include information on: Gene sequencing of variety Reaction to various diseases Probable susceptibility to any abiotic or biotic stress Information on genes to be altered to remove defects Can be used for patenting Developer rights can be verified (name of developer included)

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APPLICATIONS OF SEQUENCE INFORMATION IN PLANT GENOME ANALYSIS Classical And Advanced Approaches EST database through sequence information

Genotyping-by-Sequencing in Plants:

Genotyping-by-Sequencing in Plants Genotyping by sequencing (GBS) is a simple highly-multiplexed system for constructing reduced representation libraries for the Illumina next-generation sequencing platform. Key components of this system are: reduced sample handling, fewer PCR and purification steps, no size fractionation and inexpensive barcoding. We use restriction enzymes to reduce genome complexity and avoid the repetitive fraction of the genome.

Genotyping-by-Sequencing in Plants:

Genotyping-by-Sequencing in Plants Genotype by sequencing can be used in plant breeding by various approaches. By using various breeding populations like F2 , RILs , NILs . We can develop further refine biparental populations by fine mapping of molecular markers. These biparental populations are then use to tag a particular gene and preparations of genetic and linkage maps. If association analysis is known for a set of economically viable characters . These physical maps can be use to fish out genes and further can be used to develop transgenics .

Genotyping-by-Sequencing in Plants :

Genotyping-by-Sequencing in Plants The advent of next-generation DNA sequencing (NGS) technologies has led to the development of rapid genome-wide Single Nucleotide Polymorphism (SNP) detection applications in various plant species. Recent improvements in sequencing throughput combined with an overall decrease in costs per gigabase of sequence is allowing NGS to be applied to not only the evaluation of small subsets of parental inbred lines, but also the mapping and characterization of traits of interest in much larger populations. Such an approach, where sequences are used simultaneously to detect and score SNPs, therefore bypassing the entire marker assay development stage, is known as genotyping-by-sequencing (GBS). This review will summarize the current state of GBS in plants and the promises it holds as a genome-wide genotyping application.

Sequencing applications in development of molecular marker:

Sequencing applications in development of molecular marker RAPD- primer synthesis RFLP- probe designing AFLP- primers designing SSR- primer designing SNP- detection CAPS- primer designing EST- primer designing RGA - probe designing FISH- Probe designing Mol. Marker sequence information used for

Other marker techniques :

Other marker techniques Amplified fragment length polymorphisms (AFLPs). Sequence characterized amplified region (SCARs). Cleaved amplified polymorphic sequences (CAPS). Intersimple Sequence Repeats (ISSRs). Direct amplification of length polymorphism (DALP).

SNPs an important tool for genome analysis:

SNPs an important tool for genome analysis The analysis of genomic variation is an essential part of plant genetics and crop improvement. Use of genotyping has enabled the characterization and mapping of genes and metabolic pathways in plants as well as the study of species diversity and evolution, marker-assisted selection (MAS), germplasm characterization and seed purity. Single Nucleotide Polymorphisms (SNPs) have emerged as the most widely used genotyping markers due to their abundance in the genome.

SSR Class II DNA Markers:

SSR Class II DNA Markers Class II SSRs were estimated to occur every 3.7 kb in BAC ends and every 1.9 kb in fully sequenced BAC and PAC clones. GC-rich trinucleotide repeats (TNRs) were most abundant in protein-coding portions of ESTs and in fully sequenced BACs and PACs, whereas AT-rich TNRs showed no such preference, and di- and tetranucleotide repeats were most frequently found in noncoding, intergenic regions of the rice genome. (potentially variable markers)=SSRs ≥12 bp <20 bp.

Ultra-high Throughput Genotyping Applications :

Ultra-high Throughput Genotyping Applications Plant phylogenetic relationships Diversity studies QTL mapping. MAS( MARKER ASSISTED SELECTION)

Ultra High Throughput DNA Sequencing:

Ultra High Throughput DNA Sequencing COMPARATIVE GENOMICS HUMAN BEINGS CHIMANZEE MOUSE RICE WHEAT MONKEY

Applications of NGS Technologies to Genotyping-by-Sequencing:

Applications of NGS Technologies to Genotyping-by-Sequencing The development of markers, as well as their scoring across populations. BLAST SNP Detection

Applications of NGS Technologies to Genotyping-by-Sequencing:

Applications of NGS Technologies to Genotyping-by-Sequencing Repetitive sequences( In maize or wheat much of the sequence is repetitive) Ploidy Presence or absence of homeologs. Whole genome resequencing

strategies for reducing the complexity of a genome:

strategies for reducing the complexity of a genome mRNA-6HT´ strategy cDNA molecules are chemically cleaved and the resulting fragments are end-sequenced. Distinct methylation Use of methylation-sensitive restriction endonucleases to enrich for low-copy hypomethylated regions of the genome. Other strategies Multiplexed amplification of target sequences Molecular inversion probes (MIPs) The use of probes to capture DNA fragments by direct hybridization prior to sequencing are available but can be labor intensive and tough .

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CASE STUDIES

Genome Analysis in Arabidopsis thaliana :

Genome Analysis in Arabidopsis thaliana Arabidopsis thaliana is the first plant for which the complete genome has been sequenced and published. It is a small plant in the mustard family . The 120-megabase genome of Arabidopsis is organized into five chromosomes and contains an estimated 20,000 genes. More than 30 megabases of annotated genomic sequence has already been deposited in GenBank by a consortium of laboratories in Europe, Japan, and the United States.

Analysis of the genome sequence of Arabidopsis thaliana :

Analysis of the genome sequence of Arabidopsis thaliana Is a model system for identifying genes and determining their functions. The sequenced regions cover 115.4 megabases of the 125 -megabase genome and extend into centromeric regions. Arabidopsis has many families of new proteins but also lacks several common protein families, indicating that the sets of common proteins have undergone differential expansion and contraction in the three multicellular eukaryotes. The evolution of Arabidopsis involved a whole-genome duplication, followed by subsequent gene loss and extensive local gene duplications, giving rise to a dynamic genome enriched by lateral gene transfer from a cyanobacterial . The genome contains 25,498 genes encoding proteins from 11,000 family

Analysis of the genome sequence of Pseudomonas putida:

Analysis of the genome sequence of Pseudomonas putida Pseudomonas putida is a saprophytic soil bacterium. Is a biosafety host for the cloning of foreign genes. It has considerable potential for biotechnological applications. Sequence analysis of the 6.18  Mb genome of strain KT2440 reveals diverse transport and metabolic systems . It has high level of genome conservation with the pathogenic Pseudomonad Pseudomonas aeruginosa. Analysis of the genome gives the non-pathogenic nature of P.putida .

Analysis of the genome sequence of Rice (Oryza sativa ) :

Analysis of the genome sequence of Rice ( Oryza sativa ) DNA sequence used to determine the frequency and distribution of different simple sequence repeats (SSRs) in the genome. SSR Class I (hypervariable markers)=SSRs ≥20 bp Class I SSRs in end-sequences of Eco RI- and Hin dIII-digested BAC clones was one SSR per 40 Kb. In continuous genomic sequence (represented by 27 fully sequenced BAC and PAC clones), the frequency was one SSR every 16 kb. A set of 200 Class I SSR markers was developed. This contribution brings the number of microsatellite markers that have been rigorously evaluated for amplification, map position, and allelic diversity in Oryza spp. to a total of 500 .

Analysis of the genome sequence of Munich :

Analysis of the genome sequence of Munich To develop an integrated cross-species plant genome resource, we maintain comprehensive databases for model plant genomes, including Arabidopsis ( Arabidopsis thaliana ), maize ( Zea mays ), Medicago truncatula , and rice ( Oryza sativa ). The Munich Information Center for Protein Sequences (MIPS) has been involved in maintaining plant genome databases since the Arabidopsis ( Arabidopsis thaliana ) genome project ( Arabidopsis Genome Initiative, 2000 ).

Analysis of the genome sequence of Munich :

Analysis of the genome sequence of Munich Arabidopsis data is available from several genome databases, including The Arabidopsis Information Resource (TAIR) The Institute for Genomic Research (TIGR)and the MIPS Arabidopsis thaliana Database (MAtDB). Rice ( Oryza sativa ) is available through TIGR, Oryzabase or Gramene and maize ( Zea mays ) is maintained at MIPS or MaizeGDB .

To overcome these limitations, an integrated cross-species plant genome resource is desirable . The challenges are as follows. :

To overcome these limitations, an integrated cross-species plant genome resource is desirable . The challenges are as follows. Maintaining current and comprehensive data on the most relevant plant model genomes. Standardization of data representations and interoperability with primary databases can significantly ease the task of data integration and updates. Genome analysis previously focused on the protein coding genes, whereas recently, noncoding elements such as microRNAs have received intense attention . The ability to work with heterogeneous data sets. Limitations and methods to overcome them

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DE NOVO APPROACH

De Novo Next Generation Sequencing of Plant Genomes :

De Novo Next Generation Sequencing of Plant Genomes Next generation sequencing technologies have brought about great improvements in sequencing throughput and cost, but do not yet allow for de novo sequencing of large repetitive genomes as found in most crop plants.

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OTHER APPLICATIONS

Yeast microarrays for genome wide parallel genetic and gene expression analysis :

Yeast microarrays for genome wide parallel genetic and gene expression analysis High-density DNA microarrays of yeast ORFs. These microarrays can monitor hybridization to ORFs for applications such as quantitative differential gene expression analysis and screening for sequence polymorphisms. Automated scripts retrieved sequence information from public databases to locate predicted ORFs and select appropriate primers for amplification. The primers were used to amplify yeast ORFs in 96 -well plates, and the resulting products were arrayed using an automated micro arraying device.

Yeast microarrays for genome wide parallel genetic and gene expression analysis :

Yeast microarrays for genome wide parallel genetic and gene expression analysis Arrays containing up to 2,479 yeast ORFs were printed on a single slide. The hybridization of fluorescently labeled samples to the array were detected and quantitated with a laser confocal scanning microscope. Application Genetic and gene expression analysis at the whole genome level.

Crop genomics: advances and applications :

Crop genomics: advances and applications The completion of reference genome sequences for many important crops and the ability to perform high-throughput resequencing are providing opportunities for improving our understanding of the history of plant domestication and to accelerate crop improvement. Crop plant comparative genomics is being transformed by these data and a new generation of experimental and computational approaches. The future of crop improvement will be centred on comparisons of individual plant genomes, and some of the best opportunities may lie in using combinations of new genetic mapping strategies and evolutionary analyses to direct and optimize the discovery and use of genetic variation. Here we review such strategies and insights that are emerging.

Conclusions:

Conclusions Gene sequencing has been used widely in a range of applications like Forensic science, Diagnostics, studies, Medicines and Agriculture . Agricultural applications include fingerprinting, p atenting, registration of a genotype, SNP detection, Primer designing, Mutant Detection, QTL analysis, Biotechnology. Genome sequencing, and Genome data cards.

Conclusions:

Conclusions High-throughput variant discovery has been made possible in multiple species by the recent advent of next-generation DNA sequencing technologies. Continuous increase in sequencing throughput and the accompanying decrease in consumable cost per Gbp has allowed researchers to switch focus from resequencing small panels of parental individuals for the sole purpose of discovering variants to resequencing much larger pools of individuals within a population, where the sequenced differences are used directly as genotypic markers.

Conclusions:

Conclusions This genotyping-by-sequencing (GBS) approach has several advantages, including the facts that no preliminary sequence information is required and that all newly discovered markers originate from the population being genotyped.

Conclusions:

Conclusions It is expected that, as the amount and quality of sequencing information generated per run keeps increasing, thus allowing even higher plexing and lower costs per samples, plant breeders soon may be able to sequence even larger populations, allowing genomic selection or the determination of a population structure without prior knowledge of the diversity present in the species .

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APPLICATIONS OF SEQUENCE INFORMATION IN PLANT GENOME ANALYSIS Classical And Advanced Approaches

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