NGS Sequencing Services
Library Preparation: An NGS library is made up of random fragments that represent the entire sample. It is created by shearing DNA into 150-400 base fragments. These fragments are ligated to specific adapters. Library fragments of the appropriate size are then selected (size is application dependent) and isolated. Following a sample cleanup step, the resultant library is quantified by qPCR and checked for quality using the Agilent TapeStation. The SMF has automated library preparation for most applications using the Eppendorf EPMotion 5075 and Agilent Bravo Liquid Handlers.
Cluster Generation: Library fragments are bound to a flow cell by hybridizing the fragments to a lawn of oligonucleotides complementary to the adapter sequences. Bound fragments are clonally amplified by bridge amplification to create millions of individual dense clusters of clones. Cluster generation occurs in a closed environment on the Illumina cBOT instrument. Cluster generation occurs on-instrument on the NovaSeq6000 and NextSeq500 instruments.
Illumina HiSeq Sequencing: Sequencing on the flow cell employs Illumina’s well-established sequencing-by-synthesis chemistry. This chemistry utilizes two (NovaSeq6000, NextSeq500) or four (HiSeq2000, HiSeq4000 and MiSeq) reversible terminator nucleotides, each possessing a chemically blocked hydroxyl group. To begin sequencing, primers are hybridized to single stranded, covalently bound templates on the flow cell. Fluorescently labeled nucleotides are then flowed across the flow cell. During chain extension the fluorescent nucleotides compete for incorporation into th growing DNA chain. A single complementary nucleotide is incorporated into each DNA, terminating the chain and resulting in the simultaneous one base extension of millions of DNA clusters. The incorporated nucleotides are excited by a laser, and emit their characteristic fluorescence (or lack of fluorescence). This fluorescence is detected and recorded in an imaging step. Following base detection, the fluorescent dye is cleaved and the 3’ hydroxyl block is chemically reversed, allowing chain extension to continue. This is repeated 36 to 300 times, generating a series of images.
For consulting services, please email at nnavin (at) mdanderson.org.