L5_DNA SEQUENCING APPROACH

DNA Sequencing Techniques

Page 1: Basics of DNA Sequencing

  • DNA Sample: SCAC GOGGGGCCAGCTG

  • Protein Sequence: PRO CCCTC

  • Sample Sequences: Various sequences highlighting DNA variability

Page 2: Overview of Sequencing Techniques

  • Direct Sequencing of PCR Product

  • Genome Sequencing Approaches:

    • Hierarchical Shotgun Sequencing

    • Whole Genome Shotgun Sequencing

  • Single End and Paired End Sequencing

Page 3: Direct Sequencing of PCR Product

  • Description: Direct sequencing of amplified DNA focusing on specific target genes.

  • Data Quality: High-quality sequencing data demand strong, specific PCR product.

  • Advantages:

    • Eliminates time-consuming cloning procedures.

    • Avoids repetitive operations like template extraction.

  • Applications:

    • Gene mutation detection

    • Genetic disease diagnosis

    • Single nucleotide polymorphism research

    • Microorganism species identification

  • Steps:

    1. Extract DNA from samples.

    2. Perform PCR on target genes.

    3. Purify PCR product.

    4. Conduct sequencing.

Page 4: Genome Sequencing Approaches

  • Hierarchical Shotgun Sequencing:

    • Creates a physical map of the genome before sequencing.

    • Involves large pieces of DNA analysis.

  • Whole Genome Shotgun Sequencing:

    • Randomly shears entire genome into small fragments.

    • No need for a physical map; relies on computer assembly.

  • Factors Determining Sequencing Strategy:

    • Genome size

    • Chromosomal structure

    • Repeat content and characteristics

Page 5: Hierarchical Shotgun Sequencing Details

  • Also Known As:

    • BAC-by-BAC or clone-by-clone strategy

    • Map-based method

  • Applicability: Useful for higher-order vertebrate genomes with repetitive sequences.

  • Process:

    1. DNA mapped with genetic markers.

    2. Cut into sub-clones aligned by markers.

    3. Fragments sequenced, matched based on overlaps.

    4. Construct DNA contig from sequenced sub-clones.

Page 6: Generating a Physical Map

  • Library Creation:

    • Fragment target genome and insert into BAC vectors.

    • Transform into E. coli to replicate fragments.

  • Fingerprinting:

    • Utilize restriction enzymes for clone fingerprinting.

    • Identify overlapping clones to define genetic structure.

  • Sequencing:

    • Fragment larger clones before individual sequencing.

    • Assemble shotgun sequences for genome.

Page 7: Hierarchical Shotgun Sequencing Utilization

  • Constructs genetic and physical maps from diverse sequence data.

  • Serves as landmarks on developing genomic physical maps.

Page 8: Whole Genome Shotgun Sequencing

  • Process: Sequence numerous overlapping DNA fragments simultaneously.

  • Mechanics: Utilizes computer programs to identify overlaps and assemble sequences.

  • Usage: Common for sequencing microbial genomes due to smaller genome size.

Page 9: Comparison of Sequencing Approaches

  • Advantages of Hierarchical Shotgun:

    • Higher accuracy due to known chromosomal locations.

    • Faster and cost-effective.

  • Disadvantages of Whole Genome Shotgun:

    • More error-prone due to random assembly.

    • Simpler process with fewer assembly steps.

Page 10: Single and Paired End Sequencing

  • Single-end Read Sequencing: Sequences DNA from one end only.

  • Paired-end Read Sequencing:

    • Sequences both ends; better alignment data.

    • Advantages include higher read quality and ability to detect indels.

Page 11: Benefits of Paired-end Reading

  • Improves identification of read positions in the genome.

  • More effective for resolving structural rearrangements and assembling repetitive regions.

Page 12: Genome Assembly Process

  • Definition: Assembly of DNA fragments to reconstruct complete genome sequence.

  • Approaches:

    • De novo Assembly

    • Reference-guided (resequencing) Assembly

Page 13: De Novo Assembly Explained

  • Purpose: Used when no reference genome exists.

  • Process:

    • Assemble short reads into longer contigs.

Page 14: Sequencing and Reconstruction

  • Sequencing: Genome fragmentation followed by individual sequencing of short reads.

  • Error Correction: Correction of errors particularly in long-read technologies.

  • Overlap Identification:

    • Three algorithms: OLC, De Bruijn graphs, string graphs.

    • Merging reads to form contigs, ordered into scaffolds.

Page 15: Applications and Challenges of De Novo Assembly

  • Applications: Assembling genomes without reference or new reference-quality assemblies.

  • Challenges: Computational intensity and issues with repetitive genome areas.

Page 16: Conclusion

  • Acknowledgment: Thank you.