Genome sequencing,shotgun sequencing.pptx

Genome sequencing,shotgun sequencing.pptx

1. Genome Sequencing, Shot gun Sequencing

2. Genome : Complete set of genetic information from a haploid set of chromosomes of an organism. Genome is found in every cell, inside the nucleus. Genomics: The study of structure and function of whole genomes and their interactions rather than single genes or proteins. INTRODUCTION

3. The sequence information of genome will show The position of every gene along the chromosome The regulatory regions that flank each gene The coding sequence that determines the protein produced by each gene Genome sequencing is the process of determining the complete DNA sequence of an organism's genome. Sequencing involves determining the order of bases. Importance: Essential for understanding genetic makeup, evolution, and disease.

4. Collecting a sample containing the DNA of interest. This could be a blood sample, tissue biopsy, saliva, or any other biological material depending on the organism being studied . DNA Shearing Sample Collection: DNA Extraction: Scientists cut the DNA into short fragments of known length, either by using enzymes or mechanical disruption DNA Library Preparation Scientists make many copies of each DNA fragment using a process called polymerase chain reaction (PCR) This is typically done using various biochemical methods that break open the cells and separate the DNA from other cellular components such as proteins and lipids. Basic steps in genome sequencing

5. Basic steps in genome sequencing Sequencing The prepared DNA fragments are then loaded onto a sequencing platform where they undergo a series of biochemical reactions to determine their nucleotide sequence. There are several sequencing technologies available, including Sanger sequencing, Illumina sequencing Genome Annotation After the genome sequence is assembled, it needs to be annotated to other functional elements within the genome. This involves predicting gene locations, identifying coding and non-coding regions such as promoters, enhancers, and repetitive sequences. DNA Sequence Analysis The sequencer produces millions of DNA reads, and specialized computer programs are used to put them together in the correct order. Validation This can be done using various experimental techniques such as polymerase chain reaction (PCR), Sanger sequencing, and functional assays to confirm the presence and functionality of predicted genes and regulatory elements.

6. Determining the sequence of DNA Methods Maxam and Gilbert chemical degradation method Chain termination or deoxy method Frederick Sanger method Genome sequencing method: Shotgun sequencing Second generation sequencing methods: Pyro sequencing Nanopore sequencing Illumina sequencing Solid sequencing

7. Factors determining sequencing strategy Genome size Chromosomal structure Repeat condensed character Desired end product

8. Shotgun sequencing It is a method used in molecular biology to determine the DNA sequence of an organism's genome. It is called " shotgun " sequencing because it involves randomly breaking up DNA sequences into lots of small pieces and then reassembling the sequence by looking for regions of overlap. Originally used by Sanger and his colleagues to sequence small genomes such as those of viruses and bacteria.

9. History 1970s: Early DNA Sequencing Techniques ( Frederick Sanger ) 1980s: Initial Concepts of Shotgun Sequencing 1990s: Shotgun Sequencing of Small Genomes ( Bacteria, Virus ) Notable achievements include the sequencing of the bacterium Haemophilus influenzae in 1995, the first complete genome sequence of a free-living organism. 2000s: Human Genome Project and Next-Generation Sequencing 2005: Rapid Advances in NGS Technologies

10. Principle of shotgun sequencing The chain termination method of DNA sequencing can only be used for fairly short strands of 100 to 1000 base pairs. The principle of shotgun sequencing involves breaking the genome into small, random fragments, sequencing these fragments, and then assembling them back together to reconstruct the original genome sequence.

11. TWO METHODS IN GENOME SEQUENCING Heirarchial shot gun sequencing Useful for sequencing genomes of higher vertebrates that contain repetitive sequences Whole genome sequencing It is useful for smaller genome

12. Hierarchical shotgun sequencing Imagine a jigsaw puzzle with billions of tiny pieces, that's the challenge of sequencing DNA. Hierarchical shotgun sequencing breaks down this complexity by breaking DNA into manageable fragments

13. Hierarchical genome sequencing It is a method of DNA sequencing that involves breaking down the genome into smaller, more manageable fragments, sequencing those fragments, and then assembling the sequence data to reconstruct the entire genome in a hierarchical manner. Principle Break the genome into manageable fragments. Sequence each fragment. Assemble the sequence data to reconstruct the genome.

14. The genome is initially fragmented into manageable pieces using physical or enzymatic methods. Fragments are cloned into vectors, such as bacterial artificial chromosomes (BACs) or yeast artificial chromosomes (YACs), to generate larger insert clones. Cloning helps in handling larger DNA fragments that are difficult to sequence directly. The cloned fragments are further subcloned into smaller inserts to facilitate sequencing. This step generates subclones with insert sizes suitable for sequencing Fragmentation Cloning Subcloning Steps

15. Steps Shotgun Sequencing Each subclone is sequenced individually using high-throughput sequencing technologies, such as Sanger sequencing or next-generation sequencing platforms. This step generates sequence reads for each subclone. Integration Contigs are integrated to reconstruct the entire genome sequence. This integration involves merging overlapping contigs and filling gaps between them to generate a comprehensive genome assembly Assembly Computational algorithms are used to assemble the sequence reads from individual subclones into contiguous sequences called contigs . Contigs represent larger portions of the genome and are assembled hierarchically based on overlapping sequences from subclones.

16. Advantages and Disadvantages Allows for sequencing larger genomes by breaking them into manageable, larger pieces initially. Reduces the complexity of sequence assembly by sequencing and assembling larger fragments first. Can be more cost-effective for sequencing large genomes compared to whole genome shotgun sequencing. Advantages Requires additional steps for fragmenting and sequencing larger DNA segments, which can increase the overall time and cost of sequencing. May miss smaller genetic variations or sequences present within the larger fragments if not adequately covered during sequencing Disadvantages

17. Whole genome Sequencing In this approach, the genome is decomposed directly into individual random reads. Then attempts to assemble the genome as a whole. Principle: Randomly fragment the genomic DNA into small, overlapping fragments. Sequence the fragments using high-throughput sequencing technologies. Assemble the sequence reads into contiguous sequences using computational algorithms.

18. Steps Isolation of Genomic DNA: The process begins with the isolation of genomic DNA from the organism of interest. Random Fragmentation of Genomic DNA: The isolated genomic DNA is then randomly fragmented into small pieces. This can be achieved through methods such as sonication or nebulization. Size Selection Using Electrophoresis: The fragmented DNA is subjected to size selection using electrophoresis, which allows for the isolation of DNA fragments of the desired size range.

19. Steps Library Construction: This involves the ligation of sequencing adapters to the DNA fragments, which are essential for the subsequent steps of the sequencing process. Paired-End Sequencing (PE Sequencing): The DNA library is subjected to paired-end sequencing, which involves sequencing both ends of the DNA fragments. This step provides crucial information about the relative positions and orientations of the DNA fragments within the genome. Genome Assembly: This process involves using specialized computer programs to align and merge the individual DNA reads to reconstruct the original genome sequence. The overlapping sequences are used to piece together the complete genome.

20. Advantages Provides a comprehensive and unbiased view of the entire genome, including smaller genetic variations and sequences. Can be more suitable for sequencing smaller genomes or genomes with less repetitive regions. Offers flexibility in sample preparation and sequencing, allowing for faster turnaround times and lower costs compared to hierarchical shotgun sequencing Disadvantages Requires more computational resources and sophisticated algorithms for assembling the fragmented sequences accurately. May encounter challenges in assembling repetitive regions or resolving complex genomic structures.

21. Applications Large Genome sequencing : Used in sequencing large and complex genomes. Example: Sequencing of maize genome , Human genome project Metagenomics: Used to study microbial communities in environmental sample Example: Global ocean sampling expedition. Cancer Genomics: Used to identify genetic mutations and alterations in cancer cells Example: The Cancer Genome Atlas project Ancient DNA Studies: Shotgun sequencing has been crucial in studying ancient DNA from archaeological samples.

22. References Ranjan, R., Rani, A., Metwally , A., McGee, H. S., & Perkins, D. L. (2016). Analysis of the microbiome: Advantages of whole genome shotgun versus 16S amplicon sequencing. Biochemical and Biophysical Research Communications, 469(4), 967–977. Marra, M. (2024). Whole genome sequencing, genetics. The Editors of Encyclopaedia Britannica . Waterston, R. H., Lander, E. S., & Sulston, J. E. (2002). On the sequencing of the human genome. Proceedings of the National Academy of Sciences, 99(6), 3712-3716. Koskinen, V. R., Emery, P. A., Creasy, D. M., & Cottrell, J. S. (2011). Hierarchical clustering of shotgun proteomics data . Molecular & Cellular Proteomics, 10(6). https://www.bio.davidson.edu/courses/genomics/method/shotgun.html https://www.news-medical.net/life-sciences/Shotgun-Sequencing.aspx