ABSTRACT
This chapter presents a slightly simplified version of the Illumina technology for next-generation sequencing (NGS). The library preparation ("library prep") step creates a collection ("library") of random DNA fragments that are ready to be sequenced. There are four major substeps for library prep: Fragment DNA, Repair ends and add A overhang, Ligate adapters, Select ligated DNA. To generate the adapter fork, both adapters are combined with a buffer and the solution is heated and then allowed to slowly cool down. The purpose of enrichment PCR is to selectively enrich those DNA fragments that have adapter molecules on both ends and to amplify the total amount of DNA in the library. A flow cell is essentially a hollow glass slide. It can have one or multiple channels ("lanes"). A base-calling algorithm assigns (calls) bases and associated quality values for each position of the read. The quality of the sequenced bases is assessed by Illumina sequencers with a procedure called chastity filter.
