Genome Sequencing Techniques: Traditional and Next-Generation
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Genome Sequencing Techniques: Traditional and Next-Generation

Whole-genome sequencing is the main approach to determining the sequence of massive segments of DNA into smaller pieces. The Traditional Whole-Genome Shotgun Sequencing is similar to Next-Generation Whole-Genome Shotgun Sequencing, but the next-generation technique is much more efficient in producing substantial amounts of sequencing reactions simultaneously. In this article, you will be able to understand how the two approaches to whole-genome sequencing in a more simpler term and which one is more popular today.

The genome project began when a group of researchers decided to sequence and clone the whole genome of a specific organism. The purpose of sequencing whole entire genomes is to analyze how the genetic information can affect the physiology and development of living organisms. In addition, this can also be used to identify any genes that may be harmful to the organism such as a disease.  The human genome has the highest resolution map which consists of 3 billion base pairs (Adenine, Guanine, Thymine, Cytosine) of DNA. In order to sequence a genome, the procedures include cutting up the DNA into thousands to millions of pieces and overlapping the smaller segments. Next, each small segment in the sequence is read and the overlap where the segments are identical are obtained. Then, continue to overlap larger segments until all the smaller segments are linked. As a result, the genome is sequenced. Unfortunately, with billions of nucleotides to sequence, a sequencing machine does not exist. However, depending on the length of the DNA sequence, there are certain automated sequencing methods that can be used based on speed, cost, and accuracy. There are two methods of sequencing, Traditional Whole Shotgun Sequencing (WGS) and Next-Generation WGS.

In Traditional WGS, the short segments of DNA that makes up the genomic library are inserted into an accessory chromosome. The process of constructing a genomic library first consists of using restriction enzymes to cleave and cut purified DNA into shorter segments. Then, the fragments are joined by DNA ligase to the complementary fragment of the accessory chromosome. By inserting the DNA into bacterial cells, each cell is occupied with one recombinant molecule. The recombinant molecule undergoes replication and division allowing the molecule to be amplified from a cell that is a clone. These randomly selected clones are obtained from a shotgun library because the sequence reads are randomly selected from the whole-genome library with no information on where these are located in the genome. To sequence the genome fragments, a primer from each end of the vector is used to produce short random segments in opposing direction, with some segments overlapping. Thus producing a large library of short sequence reads covers the whole genome by matching homologous sequences with overlapping ones. Finally, these overlapping sequences form into units known as sequence contigs. 

In Next-generation whole-genome shotgun sequencing, the goal is similar to the traditional WGS however, the methods used in next-generation is dramatically distinct from the traditional WGS. One of the most commonly used method in next-generation WGS has three stages. In the first stage,  single-stranded DNA molecules are formed, making the DNA template library. In the second stage, the DNA molecules are amplified into multiple copies through a technique called Polymerase Chain Reaction (PCR). First, the single DNA strands are immobilized on each bead and amplified by PCR where the the DNA is still attached to the bead containing many identical DNA. Then each bead is inserted into a tiny well. In the third stage, each bead is sequenced by a technique called pyrosequencing. By adding DNA polymerase and a primer to the wells, you can synthesize a complementary DNA. In a particular order, every one of the four deoxyribonucleotides dATP, dGTP, dTTP and dCTP is supposed to travel through the wells one at a time. Then a pyrophosphate molecule is released when a nucleotide is added to the complementary base in the template strand. In order to transform the pyrophosphate signal into a visible light signal, two enzymes (sulfurylase and luciferase). After this process is repeated for at least 100 cycles, the signals from each well constructs the overall sequence reads. Surprisingly, using this method can substantially sequence hundreds of thousands to millions of reactions at the same time.

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