Abstract
Clinical genetics has an important role in the healthcare system to provide a definitive diagnosis for many rare syndromes. It also can have an influence over genetics prevention, disease prognosis and assisting the selection of the best options of care/treatment for patients. Next-generation sequencing (NGS) has transformed clinical genetics making possible to analyze hundreds of genes at an unprecedented speed and at a lower price when comparing to conventional Sanger sequencing. Despite the growing literature concerning NGS in a clinical setting, this review aims to fill the gap that exists among (bio)informaticians, molecular geneticists and clinicians, by presenting a general overview of the NGS technology and workflow. First, we will review the current NGS platforms, focusing on the two main platforms Illumina and Ion Torrent, and discussing the major strong points and weaknesses intrinsic to each platform. Next, the NGS analytical bioinformatic pipelines are dissected, giving some emphasis to the algorithms commonly used to generate process data and to analyze sequence variants. Finally, the main challenges around NGS bioinformatics are placed in perspective for future developments. Even with the huge achievements made in NGS technology and bioinformatics, further improvements in bioinformatic algorithms are still required to deal with complex and genetically heterogeneous disorders.
Highlights
Genetics is of extreme importance to medical practice as it provides a definitive diagnosis for many clinically heterogeneous diseases
Further technological developments marked the rising of DNA sequencing, allowing the launch of the first automated DNA sequencer (ABI PRISM AB370A) in 1986, which allowed the draft of the human genome during the following decade [5]
Every time that the polymerase incorporates the complementary nucleotide into the growing chain, a proton is released, causing pH changes in the solution, which are detected by an ion sensor incorporated on the chip
Summary
Genetics is of extreme importance to medical practice as it provides a definitive diagnosis for many clinically heterogeneous diseases. The fragments are select according to the desired library size This is limited either by the type of NGS instrument and by the specific sequencing application. In the hybrid capture method, upon the fragmentation step, the fragmented molecules are hybridized to DNA fragments complementary to the targeted regions of interest. This could be done by different methods such as microarray technology or using biotinylated oligonucleotide probes [20], which aims to physically capture and isolate the sequences of interest. Concerning the amplicon-based methods, those are based on the design of synthetic oligonucleotides (or probes), with a complementary sequence to the flanking regions of the target DNA to be sequenced. This allows that after PCR enrichment, PCR duplicates can be found by searching for non-unique fragment-UMI combinations, while the real biological duplicated will contain those UMI sequences [23,24]
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