11. Sequening techniques

Sequencing Techniques

  • Universiti Kuala Lumpur

Learning Outcomes

  • By the end of this lecture, students should be able to:

    • State the different types of DNA sequencing.

    • Explain Sanger sequencing.

    • Describe the types of Next Generation Sequencing (NGS).

    • Discuss the use of Sanger sequencing and NGS.

Background

  • Sequencing refers to the techniques used to determine the primary structure of DNA, RNA, or proteins.

  • Determining the sequence enables scientists to compare DNA/RNA/protein sequences among organisms, revealing their evolutionary relationships.

Human Genome Project (HGP)

  • Project Overview:

    • Utilized first-generation sequencing known as Sanger sequencing (chain-termination method).

    • Took 13 years, cost $3 billion, and was completed in 2003.

  • Aim of HGP:

    • Determine the order of all bases in human DNA.

    • Create gene locations maps.

    • Produce linkage maps for tracking inherited traits.

  • Outcome:

    • Successfully sequenced the entire human genome, comprising three billion base pairs.

Benefits of Human Genome Project

  • HGP has:

    • Located many disease-associated genes.

    • Led to the development of many genetic tests.

    • Improved DNA forensic technology.

    • Increased advancements in genomic studies.

    • Enhanced understanding of human evolution and migration.

History of Genome Sequencing

  • Key Milestones:

    • 1953: Discovery of DNA double helix by Watson and Crick.

    • 1970: First sequencing of DNA by Wu.

    • 1983: Development of Polymerase Chain Reaction (PCR).

    • 2001: First draft of the human genome.

    • 2009-2012: Initiation of next-generation sequencing for human whole genome studies.

Application of DNA Sequencing

  • High Throughput Sequencing (HTS):

    • Assists in understanding single-nucleotide polymorphisms (SNPs) which are common genetic variations.

    • Crucial in cancer research and tumor characterization.

DNA Sequencing Methods

  • Maxam/Gilbert chemical sequencing

  • Sanger chain termination sequencing

  • Pyrosequencing

  • Array sequencing

Description of Sanger Sequencing

  • Sequences DNA regions up to approx. 900 bp using the chain termination method.

  • Involves the incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication.

  • First commercialized by Applied Biosystems in 1986.

  • Used extensively in the HGP for sequencing small DNA fragments.

Principle of Sanger Sequencing

  1. Attach a DNA primer to denatured dsDNA.

  2. Add four solutions using heat.

  3. Electrophorese the solutions.

  4. The chains terminate at the ddNTP incorporation.

Preparation for Sanger Sequencing

  • Components Required:

    • DNA primer

    • DNA template

    • DNA polymerase

    • dNTPs (90%)

    • ddNTPs with fluorescent tags (10%)

Steps in Sanger Sequencing

  1. Heat mixture to denature the template DNA (separate strands).

  2. Reduce temperature for primer binding to single-stranded template.

  3. Raise temperature for DNA polymerase to synthesize new DNA from the primer.

  4. Enzyme extends until a ddNTP is encountered, terminating the chain.

  5. Denature just synthesized DNA into single strands.

  6. Separate ssDNA using gel electrophoresis to determine the sequence.

Detection of DNA Sequence

  • Detector produces peaks in fluorescence intensity represented in a chromatogram.

  • The DNA sequence is read from the peaks shown in the chromatogram.

Applications of Sanger Sequencing

  • Widely used in research for:

    • Targeting smaller genomic regions in numerous samples.

    • Sequencing variable regions.

    • Validating NGS results.

    • Verifying plasmid sequences and inserts.

    • HLA typing and genotyping microsatellite markers.

    • Identifying single disease-causing variants.

RNA Sequencing (RNA-seq)

  • Technique to examine the RNA sequences quantity using NGS.

  • Steps Include:

    • Isolating RNA molecules.

    • Constructing cDNA library via PCR.

    • cDNA sequencing through NGS.

    • RNA-seq analysis.

Applications of RNA-seq

  • Gene expression profiling between samples.

  • Analysis of alternative splicing events.

  • Identification of allele-specific expression and disease-associated SNPs.

  • Understanding complex biological processes and cellular diversity.

Overview of RNA-seq Technique

  • Sample interest selection and processing:

    1. Isolate RNAs.

    2. Generate cDNA, fragment, size select, add linkers.

    3. Map to genome and analyze RNA reads.

Steps in RNA-seq

  1. Isolate RNAs from selected samples.

  2. Generate cDNA library via reverse transcription and PCR amplification.

  3. Add adapters with unique barcodes.

  4. Sequence cDNAs using platforms like MiSeq and HiSeq 1500.

  5. Map cDNA sequences to reference genome and quantify expression using bioinformatics tools.

Next Generation Sequencing (NGS)

  • NGS refers to modern sequencing technologies for DNA/RNA.

  • Benefits include:

    • Requires less sample material.

    • Cost-effective and quicker processing times.

    • Useful for early diagnosis of asymptomatic individuals.

    • However, it requires complex bioinformatics for data analysis.

Applications of NGS

  • Non-human and human applications covering:

    • Epigenetics, phenotypic identification, ancestry, microbiome studies.

    • Analysis of SNPs and STRs, disease management, forensic identification.

Applications of Disease Management

  • Uses in cancer surveillance, identification of rare pathogens, and pharmacogenomics.

  • In silico diagnosis of diseases and evolutionary studies.

NGS in Malaysia

  • Overview of sequencing projects by the country and available genetic testing services outlined in data.

NGS Machines

  • Examples of platforms include:

    • MiSeq, NextSeq 500, HiSeq 2500, and others.

Library Preparation in NGS

  1. Library preparation and DNA library bridge amplification.

  2. DNA library sequencing and data collection.

  3. Analysis of contigs and assembled sequences.

454 Sequencing

  • Introduced in 2005 by Roche as the first next-generation DNA sequencer.

  • Based on pyrosequencing technology allowing high throughput.

Ion Torrent/Proton Sequencing

  • Detects hydrogen ions released during DNA polymerization.

  • Commonly used for small-scale applications.

Conclusion

  • Summary of DNA sequencing techniques and their implications in genetic research and diagnostics.