Editing Missense mutation (section)

=== Next Generation Sequencing (NGS) ===
Next Generation Sequencing (NGS) has changed the world of sequencing by decreasing the cost of sequencing and increasing the throughput.<ref>{{Cite journal |last=Chang |first=Fengqi |last2=Li |first2=Marilyn M. |date=2013-12-01 |title=Clinical application of amplicon-based next-generation sequencing in cancer |url=https://linkinghub.elsevier.com/retrieve/pii/S2210776213001427 |journal=Cancer Genetics |series=Next Generation Sequencing in Clinical Cancer Genomics |volume=206 |issue=12 |pages=413–419 |doi=10.1016/j.cancergen.2013.10.003 |issn=2210-7762|url-access=subscription }}</ref> It does this by utilizing [[Massive parallel sequencing|massively parallel sequencing]] to sequence the genome. This involves clonally amplified [[DNA]] fragments that can be spatially separated into second generation sequencing (SGS) or third generation sequencing (TGS) platforms.<ref>{{Cite book | vauthors = Xu J |title=Next-generation sequencing: current technologies and applicaitons |date=2014 |publisher=Caister academic press |isbn=978-1-908230-33-1 |location=Norfolk}}</ref> There is variation between these protocols, but the overall methods are similar. Using massively parallel sequencing allows the NGS platform to produce very large sequences in a single run.<ref name="Valencia_2013">{{Cite book | vauthors = Valencia CA, Pervaiz MA, Husami A, Qian Y, Zhang K | title = Next Generation Sequencing Technologies in Medical Genetics |date=2013 |publisher=Springer New York |isbn=978-1-4614-9031-9 |series=SpringerBriefs in Genetics |location=New York, NY |language=en |doi=10.1007/978-1-4614-9032-6}}</ref> The DNA fragments are typically separated by length using gel electrophoresis.

NGS consists of four main steps, DNA isolation, target enrichment, sequencing, and data analysis.<ref name="Valencia_2013" /> The DNA isolation step involves breaking the genomic DNA into many small fragments.<ref name="Qin_2019" /> There are many different mechanisms that can be used to accomplish this such as mechanical methods, enzymatic digestion, and more.<ref name="Qin_2019">{{cite journal | vauthors = Qin D | title = Next-generation sequencing and its clinical application | journal = Cancer Biology & Medicine | volume = 16 | issue = 1 | pages = 4–10 | date = February 2019 | pmid = 31119042 | doi = 10.20892/j.issn.2095-3941.2018.0055 | pmc = 6528456 }}</ref> This step also consists of adding adaptors to either end of the DNA fragments that are complementary to the flow cell oligos and include primer binding sites for the target DNA.<ref name="Valencia_2013" /> The target enrichment step amplifies the region of interest. This includes creating a complementary strand to the DNA fragments through hybridization to a flow cell oligo.<ref name="Valencia_2013" /> It then gets denatured and bridge amplification occurs before the reverse strand is finally washed and sequencing can occur. The sequencing step involves massive parallel sequencing of all DNA fragments simultaneously using a NGS sequencer. This information is saved and analyzed in the last step, data analysis, using bioinformatics software.<ref name="Qin_2019" /> This compares the sequences to a reference genome to align the fragments and show mutations in the targeted area of the sequence.<ref name="Qin_2019" />