Is the consistency of the NGS library size important?

[mrshope]

Hello everyone! I have a general question about the importance of keeping the library fragment size consistent between samples. All my libraries have fragments ranging from 200 to 1000 bp according to the Bioanalyzer, which is fine for the Illumina paired-end sequencing. However, some of the libraries have average size of let's say 300 bp, others - 350-400 bp. I was wondering if this could be a problem by introducing some kind of bias? As far as I can see as long as the alignment rate is the same between the different samples, there shouldn't be any other hidden source of bias. In my particular example, I am doing spiked-in RNA-seq and ChIP-seq experiments and I need to prepare libraries from sonicated genomic DNA to find out the ratio of drosophila to mouse reads in the sample to determine the consistency of the spike-in. For some reason, the sonication efficiency varies slightly from sample to sample, which results in the libraries that have slightly different fragment size distribution. I would really appreciate if you could share with me your opinion/experience/piece of advance on this matter!

[GenoMax]

In practice smaller fragments always cluster more efficiently. So you should keep that in mind. As long as your library has fragments predominantly in the 300-400 bp range this should not be a big issue but if most fragments are ~1kb then you may be biasing away from those.