# Next Generation Sequencing (NGS) The limit of NGS is often a few hundred nucleotides (100-300). However, using [[bioinformatics]], multiple sequences can be read at staggered positions and then pieced together through [[sequence alignment|alignment algorithms]]. ![[Pasted image 20240612005805.png]] ![[Pasted image 20240915222328.png|401]] --- https://www.neb.com/en-us/tools-and-resources/feature-articles/the-quantitation-question-how-does-accurate-library-quantitation-influence-sequencing https://knowledge.illumina.com/instrumentation/miseq/instrumentation-miseq-reference_material-list/000001884 ## DNA Fragment Structure ![[Pasted image 20241007121304.png]] ![[Pasted image 20240912203015.png|525]] https://www.thermofisher.com/us/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/next-generation-sequencing/dna-sequencing-preparation-illumina.html https://www.futurelearn.com/info/courses/a-practical-guide-for-sars-cov-2-whole-genome-sequencing/0/steps/338249 ## Sequencing Errors > See also: > - [[Fluorescence|Fluorescent Proteins]] **Signal Decay:** As sequencing proceeds, the fluorescent signal intensity decays with each cycle, yielding decreasing quality scores at the 3’ end of the read. This is due to: 1. Degrading fluorophores - Photobleaching The light-induced change in a chromophore, resulting in the loss of its absorption of 2. A proportion of the strands in the cluster not being elongated Therefore, the proportion of signal being emitted continues to decreaase with each cycle ## Variations of NGS