With Sanger based sequencing techniques, scientists began decoding genetic sequences from a wide array of species. Even though this was an exemplary development, the limitations of the existing methods soon become a hindrance. Next Generation Sequencing was developed to overcome these limitations and revolutionize the progress in genomic science. The idea behind Next Gen Sequencing technology is similar to Sanger based methods: the bases of a small fragment of DNA are sequentially identified after each fragment is re-synthesized from a DNA template strand. But NGS can process many reactions simultaneously instead of single or few at a time. If  NGS had been developed before the Human Genome Project, the human DNA would have been decoded in about a week for a few thousand dollars instead of 13 years and 13 billion dollars.

Applications of  Next Generation Sequencing: It is used for whole gene sequencing, target genome sequencing, resequencing, target enrichment, gene regulation, transcription analysis, epigenetic changes, metagenomics, paleogenomics etc.

Whole genome sequencing: Till few years ago, sequencing the entire genome was arduous and time consuming. With Next Generation Sequencing, large genomes can be sequenced in few days. Sequencing of genomes what have never been sequenced before (de novo sequencing) pose an interesting challenge. They have to be assembled without aligning to a reference sequence. A problem with de novo sequencing is that the short read lengths generated by NGS can lead to higher number of gaps and regions where no reads align, resulting in greater fragmentation and smaller continuous sequences which makes poorer data quality. This is usually seen in regions of the genome containing repetitive sequence elements.

Targeted Sequencing: In this technique, only the required genes or defined regions in a genome are sequenced. This approach is used to sequence large number of individuals to discover, screen and validate genetic variation within a population and to identify rare genetic variants. The two methods for making libraries for targeted sequencing projects are target enrichment and amplicon sequencing.

Next-Gen Methods:

  1. Single-molecule real-time sequencing (SMRT) is based on the sequencing by synthesis approach and allows detection of nucleotide modifications such as cytosine methylation.
  2. Ion semiconductor (Ion Torrent sequencing) has a semiconductor based detection system to the hydrogen ions that are released during the polymerization of DNA, instead of optical methods used in other methods.
  3. Pyrosequencing (454) amplifies DNA inside water droplets in an oil solution (emulsion PCR), with each droplet containing a single DNA template attached to a single primer-coated bead that forms a clonal colony. The machine contains many picoliter-volume wells each with a single bead and sequencing enzymes. Pyrosequencing uses luciferase to generate light for detection of the nucleotides.
  4. Sequencing by synthesis (Illumina) is based on reversible dye-terminators technology and engineered polymerases. DNA molecules and primers are attached on a slide and amplified with polymerase to form “DNA clusters”. Then, four types of reversible terminator bases (RT-bases) are added and non-incorporated nucleotides are washed away. A camera takes images of the fluorescently labeled nucleotides.
  5. Sequencing by ligation (SOLiD sequencing) oligonucleotides of a fixed length are labeled according to the sequenced position and then annealed and ligated by DNA ligase. The DNA is amplified by emulsion PCR. The resulting beads, each containing single copies of the same DNA molecule, are deposited on a glass slide.

 

January 17th, 2014

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