9 DNA Sequencing and Bioinformatics

• DNA sequencing is the determination of exact sequence of nucleotide bases in DNA
• Next generation sequencing is very useful and very high-tech; yields high throughput products
• Direct sequencing includes:

  • Manual sequencing
  • Chemical (Maxam-Gilbert) sequencing
  • Dideoxy (Sanger) sequencing
  • Automated fluorescent sequencing
• Chemical (Maxam-Gilbert) sequencing is developed in the late 1970s by Allan M. Maxam and Walter Gilbert

  • Requires a double- or single- stranded version of the DNA region to be sequenced, with one end radioactively labeled
  • End-labelled DNA fragments are subjected to random cleavage at adenine, cytosine, guanine, or thymine positions using specific chemical agents
• Chemical sequencing procedure:

  • We need our template which could be dsDNA or ssDNA
  • Labelled at one end of one strand with 32P (Phosphorus-32)
  • Alkaline phosphatase removes terminal P and labelled with 32P
  • Cleaving of DNA strand at specific position using chemical reaction
• Cleaved bases of Dimethyl sulfate is G only
• Cleaved bases of DMS + formic acid is A + G
• Cleaved bases of Hydrazine is C + T
• Cleaved bases of Hydrazine + NaCl is C only
• Chemical sequencing tubes:

  • First tube → DMS
  • Second tube → Formic acid
  • Third tube → Hydrazine
  • Fourth tube → Hydrazine + Salt

  
  

  Fragments are radioactive labeled
• Chemical sequencing

  • To visualize the fragments, the gel is exposed to X-ray film for autoradiography
  • A series of dark bands shows the location of radiolabeled DNA molecules
• Dideoxy (Sanger) sequencing is an enzymatic method

  • AKA Chain termination method
  • Developed by Fred Sanger in 1977
  • Widely used for sequencing individual pieces of DNA
  • Gold standard for DNA sequencing
• Dideoxy sequencing

  • Used to sequence individual pieces of DNA, such as bacterial plasmids or DNA copied in PCR
  • Gives high-quality sequence for relatively long stretches of DNA
  • Expensive and inefficient for larger-scale projects
• Automated fluorescent sequencing has chemistry same as the manual sequencing using double stranded templates and cycling sequencing.

  • 2 approaches to automated fluorescent sequencing
  • Dye primer sequencing
  • Dye terminator sequencing
  • Goal: to label the fragments synthesized during the sequencing reaction according to their terminal ddNTP
• Automated fluorescent sequencing color reactions:

  • ddATP → green dye
  • ddCTP → blue dye
  • ddGTP → black or yellow dye
  • ddTTP → red dye
• Dye terminator sequencing is performed with one of the four fluorescent dyes attached to each of the ddNTPs instead of the primer.

  • Primer is unlabeled
  • Advantage: all four sequencing reactions are performed in the same tube (or well of a plate) instead of in four separate tubes
• Pyrosequencing is included in the next generation sequencing; based on the sequencing by synthesis principle.

  • Relying on the detection of pyrophosphate release on nucleotide incorporation
  • No gels, fluorescent dyes, or ddNTP
• In pyrosequencing, every addition of nucleotides, there will be pyrophosphate ions.

  • Add dNTPS one by one
  • This will react to subtrated in the mixture and then will yield oxyluciferin.
  • The light will give the signal that the dNTPs added will be the next sequence.
• Apyrase will convert excess dntps into dndp, dnmp, and phosphate
• Next generation sequencing is a powerful platform that has enabled the sequencing of thousands to millions of DNA molecules simultaneously.

  • Ultra-high throughput (massive parallel sequencing)
  • Instruments with the capacity to sequence larger volumes of samples
  • Made sequencing much Faster and less expensive.
• We need adapter ligation for amplification in next gen sequencing
• Read means sequence of nucleotides that is sequenced
• Single-end sequencing is reading a fragment from only one strand
• Paired-end sequencing is reading a fragment from both strand
• Principle used by ROCHE 454: Pyrosequencing
• Principle used by ILLUMINA: Sequencing by synthesis
• Principle used by ABI SOLiD: Sequencing by ligation
• Principle used by ION TORRENT SYSTEMS: Ion semiconductor sequencing
• NGS technologies

  • All NGS sequencing platforms require a library obtained either by amplification or ligation with custom adaptor sequences.
  • This adaptor sequences allows for library hybridization to the sequencing chips and provide a universal priming sites for sequencing primers.
  • It facilitates in sequencing
• NGS technologies

  • Each library fragments is amplified on a solid surface such as beads or a flat silicon surface, where it will hybridize.
  • This amplification creates clusters of DNA and each originating from a single library fragment and each cluster will act as individual sequencing reaction.
  • Each machine will have its own unique cycling conditions.
• NGS technologies

  • Each machine provides raw data at the end of the sequencing run.
  • This raw data is a collection of DNA sequences generated at each clusters and this data could be further analyzed to provide more meaningful results
• Pyrosequencing has large read lengths generation

  • High reagent cost
  • High error rate over strings of 6+ homopolymers
  • e.g., Roche 454
• Roche 454 is the first NGS platform introduced in the market in 2005
• Sequencing by synthesis is utilized the step-by-step incorporation of reversibly fluorescent and terminated nucleotides for DNA sequencing

  • e.g., Illumina
• Sequencing by ligation does not utilize a DNA polymerase, instead, relies on sequencing by oligonucleotide ligation and detection.

  • Applied biosystems: SOLiD (Sequencing by Oligonucleotide Ligation and Detection)
• Ion semiconductor sequencing utilize the release of hydrogen ions during the sequencing reaction to detect the sequence of a cluster.

  • Measures the release of H+
  • More cost effective and time efficient
  • e.g., Ion torrent system
• Ion torrent system's principle is like pyrophosphate, but it measures hydrogen ions instead of pyrophosphate
• Raw output for NGS are BCL files (format)

  • Raw data is needed to be separated because machines handle massive parallel sequencing
• Bioinformatics is an interdisciplinary research area at the interface between computer and biological science

  • Involves the technology that uses computer for storage, retrieval, manipulation, and distribution of information related to biological macromolecules such as DNA, RNA, and proteins
• Database is a computerized archive used to store and organize data in such a way that information can be retrieved easily via a variety of search criteria