Abstract
Rapid advances in the development of sequencing technologies in recent years have enabled an increasing number of applications in biology and medicine. Here, we review key technical aspects of the preparation of DNA templates for sequencing, the biochemical reaction principles and assay formats underlying next-generation sequencing systems, methods for imaging and base calling, quality control, and bioinformatic approaches for sequence alignment, variant calling and assembly. We also discuss some of the most important advances that the new sequencing technologies have brought to the fields of human population genetics, human genetic history and forensic genetics.
Highlights
Determining the DNA sequence is the most comprehensive way of obtaining information about the genome of any living organism
Multicenter collaborations using numerous sequencing instruments and automated sample preparation made it possible to use Sanger sequencing in the human genome project, which took more than 10 years and US$2.7 billion to complete [3,4]
We have witnessed a rapid development of a new generation of DNA sequencing systems followed by a multitude of novel applications in biology and medicine
Summary
Determining the DNA sequence is the most comprehensive way of obtaining information about the genome of any living organism. Several novel alignment programs have been developed that are adapted to the shorter read lengths, different error distributions and larger data amounts obtained by SGS technologies (reviewed in [37]) These programs have different properties in terms of, for example, their ability to perform gapped alignment, how base qualities are used during alignment and how reads aligning to repeated regions are treated. Variant calling There are multiple programs for identifying single nucleotide polymorphisms (SNPs), copy number variations (CNVs) and structural variants between sequence reads and a reference genome (reviewed in [41]) These programs aim to distinguish authentic variants from sequencing errors and incorrect alignments by evaluating different parameters, such as base qualities, coverage and the number of reads supporting the variant. According to the current specifications of Illumina’s HiSeq2000 instrument, approximately 300 × coverage of 384 B. anthracis isolates can be obtained from a single run at a reagent cost of less than US$50 per genome
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