对二代基因测序的有些概念还是不太清楚
Illumina 平台
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3178052/
The Illumina sequencing platform is shipped with GApipeline, which implements image analysis (Firecrest), base-calling (Bustard) and alignment to reference sequences.
Illumina NGS Workflow
illumina 二代测序手册
https://www.illumina.com/content/dam/illumina-marketing/documents/products/illumina_sequencing_introduction.pdf
https://www.youtube.com/watch?v=fCd6B5HRaZ8
https://www.youtube.com/watch?v=HMyCqWhwB8E&list=UUxWMU29FF4kIG8YmQf6Zv0g
library preparation 基因文库准备
将DNA或者cDNA随机分成若干个小片段（gragmentation）
然后再将小的DNA片段与特定的Adapters相连接（Ligation）
然后就制备出了我们需要的基因文库。
Cluster Generation 生成簇
容器 ： Flow cell
视频介绍 https://www.youtube.com/watch?v=pfZp5Vgsbw0
槽内有大量的微孔， 里面都是接种了特定Adapter的基座
然后DNA在几面进行桥式扩增
最后每个微孔里面都是分别不同的DNA片段的簇（Cluster）
Sequencing 测序
测序 Illumina采用了SBS技术( Sequencing by Synthesis， 边合成边测序)
正向测序-> 逆向测序
对flowcell 进行成像
基因拼接
Data Analysis
经过基因比对之后，可以进行数据分析
- Single nucleotide polymorphism（SNP）单基因变异
- Insertion-deletion（indel）identifacation 插入鉴别
- read counting metagenomic analysis
概念介绍
base calling
base color
base call
base quality Q
probability that base call is incorrect P
Q = -10 * log10P
Base calling is the process of assigning bases (nucleobases) to chromatogram peaks. One computer program for accomplishing this job is Phred base-calling, which is a widely used basecalling software program by both academic and commercial DNA sequencing laboratories because of its high base calling accuracy.[1]
variant calling
