What Is The Minimal Sequencing Depth To Identify Long Non-Coding Rna In Cell Cultures?

Question

[wim.trypsteen]

We are planning to compare expression patterns of long non-coding RNAs among different conditions in cell culture experiments. This will be performed by transcriptome sequencing on an Illumina HISeq platform. For normal mRNAseq we usually combine up to six samples with barcoded adaptors to save costs. Yet this decreases the sequencing depth per sample. Does anyone know how many samples could be combined on a HiSeq lane to get adequate results of lncRNA expression, taking the cost of sampling into consideration?

Answer 1

[Sean Davis]

I would suggest piloting this with relatively deep sequencing to get a sense of the expression levels and relative expression between conditions. Without knowing the sequencing protocol, the lincRNAs of interest, and the sample similarities or differences, it is not easy to provide a meaningful answer. Also, keep in mind that many lincRNAs overlap known mRNAs, so you'll want to take that into account when designing your experiment.

Answer 2

[JC]

lincRNAs as all other genes are expressed in different levels, in general you can detect a few hundred lincRNAs with 20 million reads, but to detect low expressed transcripts you need to go deeper. If your intention is to compare 2 or more conditions, I think is safe to use your standard protocol for mRNA.