Answer To: Thank you so much! This is Systems Biology Report on Comparative genomics using Oxford Nanopore...
Dr Shweta answered on May 30 2022
Comparative genomics using Oxford nanopore next-generation DNA sequencing
Introduction- Comparative genomics is the field of biological research which compares with the genomic features of different organisms. The genomic features compared here are the genes, gene sequences, DNA sequences, different regulatory sequences and other genomic structural milestones. Oxford Nanopore Technologies Limited is a United Kingdom (UK)-based company which develops and sells the nanopore sequencing products that are used for the direct electronic analysis of the single molecules like DNA or RNA. The Oxford nanopore next-generation DNA sequencing is a scalable technology and with the help of this nanopore DNA sequencing reads we can study linkage and the process of phasing. It aids in understanding the de novo genome assembly and get to know how to improve the existing reference genomes, resolve the issue of complex structural variants and real time sequencing of entire microbe in single read.
It also helps in the identification of closely related species with enhanced metagenomic identification, differentiation of plasmid from the genome and exploration of epigenetic modifications with the help of direct, long-read DNA sequencing. It works by detecting the electrical signals generated when nucleic acids passed through a protein nanopore results in the change of an electrical current. The generated signals later decoded to make available the specific DNA or RNA sequence. Till the mid-2000s, the sequencing of nucleic acid (NA sequencing) was basically carried out by the Sanger’s chain termination method. Later on, due to advancing methodologies, nucleic acid sequencing was progressively supplemented by the new sequencing technologies capable to generate much larger quantities of data in a shorter period of time. Among the different technologies, long-read sequencing technologies (also known as the third-generation sequencing) can produce reads which are several kilobases in length. This development significantly improves the accuracy of genome assemblies by spanning the highly repetitive segments which poses difficulty for the second-generation short-read technologies.
Third-generation sequencing method is specifically worth for plant genomes which are tremendously large and have long stretches of highly repetitive DNA. The low base calling accuracy of the third-generation technologies required high-coverage expensive sequencing which was later followed by the computational analysis to correct all the associated errors. To minimize the associated errors, the latest long-read technologies are designed which are more accurate, less expensive and the optimal method of choice for the assembly of complex genomes. Oxford Nanopore Technologies is thus a third-generation platform used for the sequencing of native DNA strands for the generation of high-quality NA sequencing results supplemented by the new sequencing technologies capable to generate much larger quantities of data in a shorter period of time.
The Next-generation sequencing (NGS) technologies have transfigured and dominated the genome sequencing market since 2005, due to their ability to generate massive amounts of data at a faster speed. It is important for the use of NGS technologies in the field of science, medicine and technology. Since the whole genome of majority of the organisms cannot be sequenced at once due to their larger size, it is firstly broken down into small pieces and then short sequences are generated for accurate analysis of the sequence.
Later on, with the help of efficient computational approaches like the read mapping and the de novo assembly, the large amounts of data can be processed and analyzed quickly and accurately. Read mapping is the aligning process which can detects the variations in the sequenced genome in comparison to the reference genome. In the denovo assembly methods the reads were combined to construct the original sequence when a reference genome does not exist.
The major limitation of this method is the presence of short-read length repetitive regions in the genome (100–150 bp reads) that causes the errors and ambiguities for read mapping and generated the computational challenges. The main issue is that we cannot spanned the entire repetitive sequence by a single short read and these short reads further causes highly fragmented, incomplete assemblies. Thus, to solve these issues and limitations a long read that can span the entire repetitive sequence and allows continuous and complete assemblies is considered and results in the emergence of newer alternative sequencing technologies.
Nanopores are suitable for the process of sequencing since the do not require any DNA or RNA labeling or nucleotide labelling for detection during sequencing and based basically on the electronic or chemical structure of the different nucleotides present in the DNA or RNA. It allows sequencing of long reads with easy portability, low-cost input and high throughput.
Aim- The aim of this practical activity is to introduce the different tools, data types and workflow process of nanopore next-generation sequencing of genomes for analysing the various file formats used in storing reads, assessing the experimental output and for performing the quality control of the sequencing reads.
Materials and Methods
1. For wet lab experiments:
Wet lab experiments were performed to investigate the genetic determinants of antifungal drug resistance in Cryptococcus neoformans using the Oxford Nanopore next generation DNA sequencing and comparative genomics. Fluconazole susceptibility using E-test was performed to identify the mutations in C. neoformans mutant.
2. Dry lab experiments:
· For dry lab analysis the C. neoformans genomic DNA Next Generation Sequencing library was isolated and then the Whole genome sequencing, Genome assembly and variant identification and protein alignment was done using the MinION device and flow cell.
· Later, to do the FastQC analysis, the MinKNOW software was used to start the sequencing experiment and after the completion of the process the experiment was terminated.
· Now, we created a base-called sequence file which was then read with a Quality Score Reads. Files with a standard Quality Score <7 are removed from the dataset and the final output files are the FAST5 file holding the raw sequencing data and a FASTQ file holding all of the base-called sequences having an associated quality score.
Results
Wet lab results-
Cryptococcus neoformans is an obligate, anaerobic encapsulated yeast which can live in both plants and animals. These microscopic fungi, the Cryptococcus neoformans lives in the environment throughout the world and causes infection in people when it enters in their body via inhaling the surrounding air. In general, it does not cause ant major infection but some of the people who have the weakened immune system can become infected with C. neoformans and get sick from it. Generally, to kill the fungi antifungal drugs are used. Fluconazole is a well-known antifungal medication which can be used against a number of fungal infections since it has the unique fungistatic capacity or the ability to stop the growth of fungi. Fluconazole has the capacity to selective inhibit the fungal cytochrome P450 dependent enzyme- the lanosterol 14-α-demethylase. Normally this enzyme works to convert the compound lanosterol to ergosterol that is an essential component of fungal cell wall biosynthesis. When fungi get exposed to this medicine then the free nitrogen atom present the azole ring of fluconazole binds with a single iron atom present in the heme group of the enzyme lanosterol 14-α-demethylase. This selective binding prevents the oxygen activation which ultimately inhibits the...