One sample pooled in a whole lane vs multiple sample in each lane

Question

[bob]

Dear All,

I have paired-end RNA-seq data sequenced using HiSeq 4000. One sample (sample#1) out of 8 samples is pooled in a whole lane and generating much more reads number for sample#1 than all other samples.

Sample1-Replicate1-Read1 - pooled in a whole lane (lane 1) - (20GB file size, 280 million reads)
Sample1-Replicate1-Read2 - pooled in a whole lane (lane 1) - (20GB file size, 280 million reads)
Sample1-Replicate2 -R1 - lane 2 - (4GB filesize, 45 million reads)
Sample1-Replicate2 -R2 - lane 2 - (4GB filesize, 45 million reads)
Sample2-Replicate1-R1 - lane 2 - (4GB filesize, 45 million reads)
Sample2-Replicate1-R2 - lane 2 - (4GB filesize, 45 million reads)
Sample2-Replicate2-R1 - lane 2 - (4GB filesize, 45 million reads)
Sample2-Replicate2-R2 - lane 2 - (4GB filesize, 45 million reads)
Sample3-Replicate1-R1 - lane 2 - (4GB filesize, 45 million reads)
Sample3-Replicate1-R2 - lane 2 - (4GB filesize, 45 million reads)
Sample3-Replicate2-R1 - lane 2 - (4GB filesize, 45 million reads)
Sample3-Replicate2-R2 - lane 2 - (4GB filesize, 45 million reads)
Sample4-Replicate1-R1 - lane 3 - (4GB filesize, 45 million reads)
Sample4-Replicate1-R2 - lane 3 - (4GB filesize, 45 million reads)
...

What is the best way to extract the read numbers from Sample1-Replicate1 (paired-end) to the level of Sample1- Replicate#2 i.e. 45 million reads out of 280 million reads file?

Many Thanks

Answer 1

[Devon Ryan]

By "extract the read numbers" I assume you mean "compensate for the difference in sequencing depth". The answer is that edgeR/DESeq2/whatever will take care of that for you.