The entire set of an organism’s DNA is known as its genome, which includes all the coding and non-coding regions of the DNA as well. An organism’s genome contains all the information required to build and maintain that organism.
A Brief Comparison of Eukaryotic & Prokaryotic Genome
For eukaryotes, this information resides within the nucleus of the cell in the form of thread-like structures known as chromosomes, while for the prokaryotes, this information is embedded within the cytoplasm of the cell in the form of chromatin.
Eukaryotic genome is comparatively larger and complex than the prokaryotic genome. The presence of exons, introns and other regulatory elements found within the eukaryotic genome makes it complex for genomic analysis and studies.
The presence of semi-autonomous organelles, such as Mitochondria & Plastids, possess their own genetic material which makes the eukaryotic genome more complex and wider.
Since, Genome is basically the set of an organism’s entire hereditary material, i.e., DNA, so in order to analyze the genome, first we have to gather the whole genome information by sequencing it. Ergo, the first step is to ‘Sequence’ the whole genome.
To sequence a genome, it is important to know how similar it is with already sequenced & annotated genomes and to know such similarities between different genomes, various in silico approaches, tools and databases are available. One of the tools being utilized for this purpose is BLAST (Basic Local Alignment Search Tool) but to comprehend the results to extract the rational information, expert analytics are required. Same is the case with other automated tools and databases, which requires experienced analytics and professionals to analyze the genome and extract the exact and meaningful information that is required for the Genome Project we’re working on.
Types of DNA Sequencing
DNA sequencing can be divided into types:
Resequencing- Sequencing the DNA fragments having some reference Genome.
De novo- Sequencing a DNA fragment having no reference genome.
DNA Sequencing Techniques (Resequencing)
In the early 1970s, before the origination of Sanger’s sequencing, Maxam and Gilbert (in 1973) succeeded in sequencing a DNA fragment of 24 base pairs length using a sequencing technique named as “Wandering-spot analysis”, also known as “Maxam-Gilbert chemical sequencing method”, which was a time consuming and labor intensive method.
Later on, in 1977, Sir Frederick Sanger developed a new method to sequence the DNA fragments known as “Chain termination method” or “Sanger’s Sequencing”. It was a comparatively easier and less toxic method than the Maxam-Gilbert sequencing method and provides high accuracy for long sequence reads of upto 700bp. Although this method is still in use but it has some drawbacks which makes the use of this method limited since it is a time-consuming process and yields 300-1000bp long DNA fragments in a single reaction.
Since Snager’s sequencing yields upto 500bp sequenced DNA fragments, a new technique was developed to overcome this issue, which is known as “Primer walking”. This can be done by adding another primer of about 10-20bp to the reaction mixture of Sanger’s sequencing and then the next 500bp DNA fragment can be sequenced using the same DNA clone.
Another method, known as “Reversible chain terminator”, is also being used for sequencing purposes. It works on the same principle of Sanger’s sequencing but instead of elevating irreversible chain termination, it involves nucleotide incorporation in a cyclic method, fluorescence imaging and cleavage.
DNA Sequencing Techniques (De novo)
A de novo sequencing technique which can assemble a genome which hasn't yet been sequenced, is known as “Shotgun Sequencing”. It involves the analysis of DNA fragments of more than 1000bp to an entire chromosome. The basic principle for this method is generating multiple DNA fragments of various lengths from different locations of the same genome and then assembling them on the basis of their overlapping regions.
All the above mentioned techniques are relatively time-consuming and tiring. Hence, to sequence whole genomes within a shorter time with more accuracy and larger amount of data, a de novo and revolutionizing technique came into existence, known as “Next Generation Sequencing (NGS)”, which is a massively parallel sequencing technology. It allows the researchers to perform a wide variety of applications and with its ultra-high throughput, scalability and speed, it has enabled the study of biological systems at a level never before possible.
Nanopore is also a de novo sequencing technique, which is a unique, scalable technology enabling real-time analysis of long DNA fragments. The basic principle behind this method is the detection of the electric signal generated when the nucleic acids are passed through a protein nanopore, the electric signal is then decoded which provides the sequence of a specific DNA or RNA fragment.
The process of identification, measurement or comparison of genomic features such as DNA sequence, gene expression, functional or regulatory elements, structural variations and their annotations at genomic level, is known as ‘Genome Analysis’. Genome Analysis is an essential step to understand the information gathered after sequencing an entire genome or comparing two or more genomes.
Genome Analysis entails the identification of functions of the genes, gene products and their interactions. For the last few decades various advanced approaches and technologies have been devised for the functional genomic analysis, which includes revolutionizing advancements in the high-throughput approaches ranging from traditional real-time PCR reactions to more complex systems like next generation sequencing (NGS) and mass spectrometry. Not just wet lab techniques but bioinformatics tools are also required to accurate scientific results and skilled and professional analytics to understand and comprehend the results.
Possible genome analysis workflows on a newly sequenced genome:
Gene prediction: Using automated tools and human expertise, the location and functions of coding genes, in the sequence genome, can be predicted.
Genome Annotation: Genome Annotation involves 3 steps; Identification of non-coding portions of the genome, Identification of the coding (gene predicting) elements of the genome, connecting the coding elements with biological information.
Genome Assembly: It refers to the process of placing the nucleotides, or more precisely, the sequenced DNA fragments in a correct order to extract logical information from it.
Comparative Genome Analysis: It involves the comparison of two or more genomes for the identification of coding regions, regulatory elements, and gene expression of the particular genomes under study.
Genome Characterization: Also known as “Genomic profiling”, refers to a process that is being utilized to learn about all the genes and their functions in specific cell type and the interactions of the genes with each other and the environment as well.
Importance of Genome Analysis
The comprehensive and accurate functional analysis of a genome involves various fields of studies such as Proteomics, Epigenomics, Genomics, and Interactomics, which are essential for bridging the gaps in the knowledge of dynamic biological processes at cellular as well as organism level. It also involves the prediction of genes from uncharacterized genomic sequences.
Genome Analysis at BioCode & BioinfoLytics
If you’re working on any genome project and need to learn how to analyze, annotate or sequence the genome you are working on, you can join our Gray Bioinformatics plans at very affordable prices and you can be an expert in analyzing any genome, by learning from our various useful and informative tutorials on the tools and databases involved in analyzing the genomes. To join our Gray Bioinformatics plans, visit us at https://www.biocode.ltd/ and enroll yourself to develop your skills.
If you want your genome of interest to get analyzed by our experts, you can get our services from the BioinfoLytics platform, an initiative of BioCode, where we are providing our services for pairwise genome alignment, multiple genome alignment, genome analysis, gene prediction, genome-wide association analysis, genome characterization, genome database searching, genome retrieval & analysis and any analysis related to the genome.
To get the above mentioned services, all you need to do is to submit your project analysis details, submit the services charges, set the deadlines and before that deadlines our experts will provide you with the results summary and details for you to work further on your research project.
Moreover, if you’ve the expertise for such computational analysis of various genomes, we’ll be very pleased to provide you our platform of BioinfoLytics, where you can provide your services as a freelancer.
For further information on our services, visit us at https://www.biocode.ltd/bioinfolytics
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