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Human genome sequencing presents several challenges related to experimental procedures and bioinformatics. As to the first challenge, the extraction and amplification of DNA for sequencing are cumbersome procedures, which take a lot of time and require due diligence. These procedures, however, are indispensable since next-generation sequencing approaches require voluminous templates for effective sequencing of the genome. The second challenge is that the human DNA is complex because it is diploid with repetitive regions and structural variants (Mostovoy et al. 587; Bickhart et al. 643; English et al. 1).
Their existence, therefore, complicates sequencing by making the process of phasing more difficult. The production of short-reads poses a third challenge that hinders coverage of whole-genome sequencing and reduces the accuracy of the genome assembly. Huddleston et al. note that the assembly of short-reads gives rise to low-quality contigs, especially in complex regions of the human genome (688). The fourth challenge occurs because the contamination of libraries or the existence of chimeras prevents the accurate determination of the human genome (Bickhart et al. 651). The last challenge to be mentioned is that the vast volume of data generated in sequencing requires huge storage space in databases and powerful computer programs for assembly, analysis, and interpretation.
Automated Sanger sequencing is one of the novels approaches to human genome sequencing. It is regarded as the gold standard of sequencing because it is highly accurate, generates long reads, targets small regions, and is ideal for sequencing small samples. However, this approach has its setbacks: it is a tedious method that needs preparation of template DNA, and it is very slow for whole-genome sequencing. As another novel approach, sequencing by synthesis (Illumina) is an effective technology due to its accuracy, high throughput, and scalable method that generates long reads. However, its weakness is that it entails a burdensome preparation of libraries and adapters as well as the purchase of expensive equipment.
Single-Molecule Real-Time sequencing (SMRT) is a novel approach to human genome sequencing that allows real-time detection of DNA synthesis, generates long reads, is highly accurate, requires a small amount of template DNA, and does not need PCR in sample preparation. Nevertheless, SMRT has lower throughput and parallelism than that of Illumina. Nanopore MinION is another approach that proved to be advantageous because it can generate long reads of up to 200kbp as well as allows real-time analysis of sequences, is fast, affordable, and does not necessitate extensive preparation of template DNA (Jain et al. 3). Nonetheless, it has a very high error rate, and its throughput is lower in comparison to Illumina.
These approaches to human genome sequencing may be combined to augment the generation of accurate and high-quality reads. Mostovoy et al. created a hybrid approach for sequencing the human genome by combining Illumina, BioNano Genomics, and 10X Genomics-based sequencing (587). The Illumina was used to assemble short-reads while 10X Genomics-based sequencing was used to generate libraries of short-reads. Subsequently, BioNano Genomics was employed to generate physical maps and chimeric assemblies (Mostovoy et al. 588). Ultimately, the study utilized 10X Genomics in phasing and validating sequences. The hybrid approach produced not only phased but also high-quality sequences. English et al. pooled multiple sequencing technologies in evaluating structural variation in the human genome (1). Particularly, the approaches that were integrated included Illumina Nextra, BioNo Irys, short-read next-generation sequencing, and Pacific BioSciences RSII. The combined use of these approaches enhances the determination of Parliament structure and reveals structural variations in the human genome. Huddleston et al. combined single-molecule real-time- and Pacific Biosciences sequencing approaches in reconstructing complex human genomes, which improved the quality of reads and genomic sequences (688).
As an example of the utilization of the novel approaches to human genome sequencing, Nanopore MinION has been applied in sequencing the whole genome of the influenza virus. According to Wang et al., the sequencing of the influenza virus using Nanopore MinION generated sequence with an accuracy of 99% when compared to the Sanger method and Illumina MiSeq (1). Moreover, a study applied single-molecule real-time sequencing in sequencing the whole genome of the domestic goat (Bickhart et al. 643). The study combined strategies such as the assembly of long and short reads, the scaffolding of sequences, and the mapping of chromatin interactions. Single-molecule sequencing improved outcomes by about 400 times and gave the best de novo assembly of the mammalian genome. A study used Illumina in creating a hybrid approach, which employed de novo sequencing and the assembly of the human genome (Mostovoy et al. 589). The outcome of the approach gave rise to sequences that are not only phased but also of high quality.
Advancements in bioinformatics have led to the creation of algorithms that enhance storage, access, analysis, and the use of biological information, mainly genomic sequences. Since sequencing generates reads, the base-calling algorithm is essential in determining the quality and assembly of sequences as well as assessing the accuracy of scores. Phred is software that carries out base calling and assembles reads into genomic sequences. Phrap is another algorithm that assembles and aligns sequences based on the scoring matrix of similarity index. As gaps in alignment present challenges, the assignment of a higher penalty enhances the alignment of sequences. Basic Local Alignment Search Tool (BLAST) is another algorithm that aids in the identification of unknown sequences based on homology. A BLAST search gives hits, which are difficult to identify. E-values of hits are statistical values that provide the validity of sequences. Open Reading Frame Finder is an important algorithm that searches for the start- and stop-codons in six frames of sequences to identify genes. The problem with ORF Finder is that it identifies the start- and stop-codons but not genes. To overcome this challenge, open reading frames that are greater than 1kbp are considered genes.
The Sanger sequencing method has made significant contributions to the sequencing of genomes. The use of this method has been employed in sequencing whole genomes of a human (3 billion bp), a bacteriophage (5,386 bp), yeast (315,000 bp), a fruit fly (180 million bp), and a mouse (2.5 billion bp) among other organisms. Nanopore MinION improved the understanding of the structure and function of the influenza virus (Wang et al. 2015). SMRT has made a substantial impact on the study of bacteria methylomes, which elucidate the functions of methyltransferases (Murray et al. 11451). These findings have advanced the understanding of bacteria and their molecular functions. Illumina sequencing improved the sequencing of the genome of a domestic goat and offered the best contigs ever obtained from a mammal (Bickhart et al. 643). Illumina has overcome challenges associated with mammalian genomes such as repeats, diploidy, and complexity regions. Therefore, these approaches to sequencing have contributed immensely to the advancements in genomic studies.
Bickhart, Derek, et al. “Single-Molecules Sequencing and Chromatin Conformation Capture Enable De Novo Reference Assembly of the Domestic Goat Genome.” Nature Genetics, vol. 49, no. 4, 2017, pp. 643-654.
English, Adam, et al. “Assessing Structural Variation in a Personal Genome: Towards a Human Reference Diploid Genome.” BMC Genomics, vol. 16, no. 286, 2015, pp. 1-15.
Huddleston, John, et al. “Reconstructing Complex Regions of Genomes Using Long-Read Sequencing Technology.” Genome Research, vol. 24, no. 1, 2014, pp. 688-696.
Jain, Miten, et al. “The Oxford Nanopore MinION: Delivery of Nanopore Sequencing o the Genomics Community.” Genome Biology, vol. 17, no. 239, 2016, pp. 1-12.
Mostovoy, Yulia, et al. “A Hybrid Approach for De Novo Human Genome Sequence Assembly and Phasing.” Nature Methods, vol. 13, no. 7, 2016, pp. 587-591.
Murray, Iain, et al. “The Methylomes of Six Bacteria.” Nucleic Acids Research, vol. 40, no. 22, 2012, pp. 11450-11462.
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Wang, Jing, et al. “MinION Nanopore Sequencing of an Influenza Genome.” Frontiers in Microbiology, vol. 6, no. 766, 2015, pp. 1-5.