Abstract

CORRELATION APPROACH BETWEEN SHOTGUN SEQUENCING WITH DNA SEQUENCING IN MOLECULAR GENOMICS

Ashish M. Parmar, Keyur D. Patel, Nilang S. Doshi, Girish M. Kapadiya, Bhavesh S. Patel and Dr. Dhrubo Jyoti Sen*

ABSTRACT

Early efforts at sequencing the genomes of bacteria, viruses and yeast allowed scientists to test and troubleshoot methods of automated sequencing, chromosome assembly and gene annotation. Using these methods, researchers were able to sequence several unicellular genomes by the year 2000. However, many interesting biological questions remained and a great deal of these questions related to issues involving development, disease and complex traits that are found only in multicellular organisms. For example, how are limbs and tissues encoded in the genome? How is the brain made? What are the genetic contributions to behavior? Today, researchers are deeply involved in the shotgun sequencing of various complex genomes in an attempt to answer such questions. Interestingly, they have found that neither genome size nor number of genes determines the complexity of multicellular organisms. Unicellular genomes generally lack repetitive regions that are difficult to sequence and these genomes are easily assembled into chromosomes. Multicellular genomes, by contrast, are more difficult to clone, sequence and assemble. Because of the expense and slow pace associated with clone-based sequencing, researchers have mainly relied on the "shotgun" method of sequencing for multicellular genomes. The whole-genome shotgun (WGS) method entails sequencing many overlapping DNA fragments in parallel and then using a computer to assemble the small fragments into larger contigs and eventually, chromosomes. This method has the advantage of simplicity and rapidity and works best for genomes with fewer repeated regions. Genomes containing lots of repetitive sequences (like the human genome) create difficulties with chromosome assembly because the computer cannot tell which unique location to map identical DNA sequences to. The hybrid WGS method overcomes this problem by breaking the genome into overlapping clones that can also be physically mapped to the genome and then performing shotgun sequencing on these intermediate segments. The result is a large-scale map that tells the exact order for each piece of sequenced DNA. DNA sequencing helps us understand the essential genetic make-up of organisms that are studied. This is vital to help understand which species are related and which are not. Interestingly, many species which were thought to be related according to evolutionary philosophy are now being shown to be either not related or quite different and unique. DNA studies are thus showing the factual ''differentness'' of each living thing that is studied. This is contrary to the study of morphology, the study of outward appearance, which obviously can be misleading. Thus overall, these studies are another important field of study which demonstrates that some evolutionary ideas are not scientific, given that it is ultimately the DNA which determines the true character of an organism.

Keywords: DNA, Shotgun, Genome, Chromosome, Base pair, Contig, WGS, NGS, MSPA, PCR, MALDI-TOF/MS, RNAP