Big variation of output for individual library

[liaojinyue]

Hello all,

We recently received our RNA-seq data from the vendor. We sequenced 8 libraries as a pool and we were supposed to get around 30M reads for each sample. After reviewing the data, we saw uneven read number for the samples ranging from around 13M to 43M. According to the vendor, they did perform q-PCR to quantify the libraries and the explanation was that the libraries behave differently when pooled together.

So how accurately can we control the read distribution among libraries in the pool? Is it normal to see variation this big? Really appreciate if anyone can share experience.

Jason

[sara_ahmed]

It is normal to see some variability even if libraries are pooled and quantified by qPCR. That said, what you describe seems like a much larger variability than normal especially if all the libraries pooled together were generated with a similar library protocol. You tend to see more variability when mixing different library types together (small RNA libs with mRNA libs for example) because they tend to cluster differently. I would say its normal to see about 20% difference between different samples in a pool if the libraries are made the same way and quantified by qPCR.

Sara Ahmed, PhD | Director of Sequencing | Cofactor Genomics
4044 Clayton Ave | St. Louis, MO 63110 | tel. (314) 531-4647