RNA-seq Data Analysis
High-Throughput Gene Expression Assays
- Next-generation sequencing (NGS)
- Platforms:
Illumina/Solexa's Genome Analyzer, HiSeq systems, MiSeq etc.
- Applied Biosystems' SOLiD
- Roche's 454 Life Sciences
Helicos BioSciences' HeliScope
- Terminology
- Sequencing Depth or Coverage: Total number of reads mapped to the genome/transcriptome, also known as library size.
- Transcript/gene length: Number of bases in a gene.
- Read counts: Number of reads mapping to that gene/transcript (expression measurement).
- Illumina's sequencing technology
- One flow cell: 8 lanes
- One lane is often used for the control sample.
- Multiplexing:
- a way to save money by sequencing multiple samples on a single unit (an Illumina's flow cell)
- offers the exibility to construct balanced blocked designs for the purpose of testing differential expression.
- Barcoding:
- to separate inputs, can have many barcodes in a single unit
- 12 different samples can be indexed with unique subsequences and loaded onto each lane. In total, 96 samples can be sequenced per run.
- the output can be deconvoluted to individual samples.
- RNA Sequencing Pipeline