question about illumina

[found]

Hi,

I am not familiar with the experiment. I have some questions about the mRNA-Seq. hope someone can help me.
1.what is "run" means in illumina sequencing? Does it mean you separate the samples into several parts. and sequencing each part in each run? Since I noticed that in a paper, some samples have 7 runs while some have only 4 runs. Does that mean these samples have different sequencing depth.

2.why the RPKM can be used to measure the expression of one gene between two samples or tissues. RPKM is just a relative value of the read number in the total reads from a sample, it is not the absolute value of the read number. so if RPKMs for a gene from two samples are equal, but the sample 2 have more total reads. It means sample 2 also have more reads derived from the gene. Then the absolute numbr of reads from the gene is larger in sample 2 than in sample 1.

Thank you very much.

[jgibbons1]

A "run" typically means a flow cell. The GA2's have 8 flowcells (1 usually used for a control) which is why you often see "7 runs"

Concerning your RPKM question and example. It is Reads Per Kilobase per Million mapped reads, therefore it normalizes for different number of reads in each experiment. You can use this calculation across (i) transcripts of different length and (ii) experiments with different read numbers