Single-end sequencing for RNA-seq

[apredeus]

Hello everybody,

we have to run some RNA-seq experiments on HiSeq 2500, and the sequencing facility has lots of experience with exomes/genomes resequencing, but very little experience with mRNA-seq. Overall, from what I know, the consensus is that for read count-based methods (ChIP-seq, RNA-seq) you want to run single-end sequencing with shorter read length, to get more individual reads from the same reagents.

What are the pitfalls and recommendations to get the best throughput from a single-end experiment in terms of cluster density, concentrations, etc?

Thank you in advance

[kmcarr]

Hello everybody,

we have to run some RNA-seq experiments on HiSeq 2500, and the sequencing facility has lots of experience with exomes/genomes resequencing, but very little experience with mRNA-seq. Overall, from what I know, the consensus is that for read count-based methods (ChIP-seq, RNA-seq) you want to run single-end sequencing with shorter read length, to get more individual reads from the same reagents.

What are the pitfalls and recommendations to get the best throughput from a single-end experiment in terms of cluster density, concentrations, etc?

Thank you in advance

Performing a single end read will not "get more individual reads from the same reagents". The optimal cluster density (number of reads) obtained from one HiSeq 2500 flow cell lane is the same regardless of whether it is a single end or paired end read, DNA, RNA or ChIP-Seq library. Once the Illumina library is made it makes (virtually) no difference to the sequencer what the starting material was or the kit used to create the library. You load an mRNA library the same way you load a DNA library. The difference is you purchase a Single Read flow cell kit and a 50 cycle reagent kit to perform your run. That's what saves you money.

[apredeus]

Thank you very much! As I don't work with the sequencer itself directly, that's what was eluding me. I knew about the "less reagents" argument, but not about the different flow cell.

Thanks again.