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This design is very different from that of Sanger sequencing—also known as capillary sequencing or first-generation sequencing—which is based on electrophoretic separation of chain-termination products produced in individual sequencing reactions. [6] This methodology allows sequencing to be completed on a larger scale. [7]
Whereas high sequence coverage for a genome may indicate the presence of repetitive sequences (and thus be masked), for a transcriptome, they may indicate abundance. In addition, unlike genome sequencing, transcriptome sequencing can be strand-specific, due to the possibility of both sense and antisense transcripts. Finally, it can be difficult ...
An important part of the design of variant calling methods using NGS data is the DNA sequence used as a reference to which the NGS reads are aligned. In human genetics studies, high quality references are available, from sources such as the HapMap project , [ 10 ] which can substantially improve the accuracy of the variant calls made by variant ...
These methods represented an important step forward in sequence assembly, as they both use algorithms to reach a global optimum instead of a local optimum. While both of these methods made progress towards better assemblies, the De Bruijn graph method has become the most popular in the age of next-generation sequencing.
Most high-throughput, next generation sequencing platforms produce shorter read lengths compared to Sanger sequencing.These new platforms are able to generate large quantities of data in short periods of time, but until methods were developed for de novo assembly of large genomes from short read sequences, Sanger sequencing remained the standard method of creating a reference genome. [10]
Formal way of sequencing, the sanger technique had some limitations that it was costly and time-consuming. The recent development of Next Generation Sequencing (NGS) [7] dramatically remedied the shortcomings of Sanger sequencing. NGS enabled the sequencing of large loads of DNA. [7] There are pieces of genes that transcribe the proteins.
RNA-Seq (named as an abbreviation of RNA sequencing) is a technique that uses next-generation sequencing to reveal the presence and quantity of RNA molecules in a biological sample, providing a snapshot of gene expression in the sample, also known as transcriptome.
Next-generation sequencing technology is performed resulting in about 100 bp single-end reads. Raw sequence data are filtered and aligned to a reference genome using usually Burrows–Wheeler alignment tool (BWA) or Bowtie 2. The next step is to identify SNPs from aligned tags and score all discovered SNPs for various coverage, depth and ...