ILLUMINA SEQUENCE.pptx
- 1. Principle and Workflow of Illumina Next-generation Sequencing Illumina, established in 1998 in San Diego, CA, is a leading company in the field of sequencing. In 2006, Illumina acquired Solexa, got the next-generation high-throughput sequencing technology and developed it into a mainstream technology on the market. It currently provides sequencing systems such as MiSeq, HiSeq 2500, HiSeq 3000, HiSeq 4000, HiSeq X Ten, HiSeq X five, NextSeq 550. Illumina NGS applications NGS has a very wide range of applications, it can be used for whole-genome sequencing, targeted region sequencing, transcriptome analysis, metagenomics, small RNA discovery, methylation profiling, and genome-wide protein-nucleic acid interaction analysis, helping people unlocking the power of the gene. The core principle of Illumina NGS The Illumina next-generation sequencing (NGS) method is based on sequencing-by-synthesis (SBS), and reversible dye- terminators that enable the identification of single bases as they are introduced into DNA strands.
- 2. The workflow of Illumina NGS Step 1. Library preparation Through ultrasonic fragmentation, the genomic DNA becomes DNA fragment with 200-500bp in length. The 5’ and 3’ adapter are added to the two ends of these small segments, “tagmentation” combines the fragmentation and ligation reactions into single step that greatly increases the efficiency of the library preparation process. Adapter-ligated fragments are then PCR amplified and gel purified. The sequencing library is constructed.
- 3. Attachment of DNA adaptor to Flow cell surface:
- 4. Unlabelled nucleotides and enzyme is added to initiate solid-phase PCR bridge amplification The enzyme incorporates nucleotides to build double-stranded bridges on the solid- phase substrate. Several million dense clusters of double-stranded DNA are generated in each channel of the flow cell. Solid-phase amplification creates up to 1,000 identical copies of each single template molecule in close proximity (diameter of 1µ or less). Denaturation leaves single-stranded templates anchored to the substrate. Low binding affinity nucleotides are washed off, only high affinity molecules remains anchored to the substrate. Bridge Amplification: Fragments become Double stranded: Denaturation of ds-DNA Complete Amplification
- 5. Step 2. Cluster generation Flow cell is a channel for adsorbing mobile DNA fragments, and it’s also a core sequencing reactor vessel — all the sequencing happens here. The DNA fragments in the sequencing library will randomly attach to the lanes on the surface of the flow cell when they pass through it. Each flow cell has 8 Lanes, each lane has a number of adapters attached to the surface, which can match the adapters added at the ends of the DNA fragment in the building process, which is why flow cell can adsorb the DNA after the building, and can support the amplification of the bridge PCR on the surface of the DNA. In theory, there is no mutual influence between these lanes. Bridge PCR was performed using the adapters on flow cell surface as template, after continuous amplification and mutation cycles, each DNA fragment will eventually be clustered in bundles at their respective locations, each containing many copies of a single DNA template. The purpose of this process is to amplify the signal intensity of the base to meet the signal requirements for sequencing. When cluster generation is complete, those templates are ready for sequencing.
- 6. The first sequencing cycle begins by adding our labelled reversible terminators, primers, and DNA polymerase. Imaging can be done using Laser emission.Similarly incorporation cycle repeated to detect 2nd base as well. After laser excitation, the emitted fluorescence from each cluster is captured and the first base is identified. Similarly the cyle is repeated to detect 2nd base and so on. The sequencing cycles are repeated to determine the sequence of bases in a fragment, one base at a time. Each raw read base has an assigned quality score so that the software used to align data can apply a weighting factor in calling differences and generating confidence scores. Step 3. Sequencing
- 7. Illumina data collection software enables users to align sequences to a reference in re-sequencing applications.This software suite includes the full range of data collection, processing, and analysis modules to streamline collection and analysis of data with minimal user intervention. The open format of the software allows easy access to data at various stages of processing and analysis using simple application program interfaces. Step 4. Alignment & Data analysis