Hey everyone,
I am trying to do a miRNA-Seq experiment. I have prepared the libraries and before sequencing, I did QC measurements on the Agilent Bioanalyzer 2100, HS DNA Kit. I have some weird results, as seen below. I need to have a peak of around 180 bp which is the sequencing library. Where another peak around 164 bp is the piRNA peak obtained. The files CDD16 & C4D19 show very high concentration peaks below 100 bp's. We thought this may be due to sample degradation, but we are not certain, wherein the protocol this degradation might have occurred, as we measured our miRNA Concentrations with the Qubit 3.0 Fluorometer, and our results were highly promising, however, our library concentrations were much lower than what we originally expected. It is not unlikely these issues are unrelated. I am aware the fluorometer might have detected degradation fragments. We are quite stumped, any opinion and suggestions for the future, on the matter will be appreciated. I have attached a different electrogram of a different sample that shows high quality, as point of reference.
I think this is very likely unincorporated oligonucleotides from the library prep. But to make the diagnosis, it would really help to have the following info:
1) What was your sample type (isolated small RNA, total RNA, or something else?)
2) How much of this sample (ng) did you put into each prep?
3) What kit did you use to prepare these libraries?
4) How many cycles of PCR did you do?
I also think you might be incorrect about the peak sizes you are looking for. Given that miRNAs are ~8nt smaller than piRNAs, it doesn't make sense that you expect the miRNA and piRNA peaks to be so far apart, or that the miRNA peak would be bigger. I have prepared a lot of libraries targeting both these small RNA types using the NEBNext kit, and this adds 126nt of sequence in total - so the expected size of the adapter dimer (if present) is 126bp, and the expected size of the library peaks is (126 + size of small RNA). Therefore, with the NEBNext kit, I'd expect to see miRNA libraries peak at 147nt (21nt insert size) and piRNA libraries around 154nt (28nt insert size). I'm fairly sure the Illumina kits produce libraries within a few base pairs of this size. The Bioanalyzer usually detects the peaks +/- about 3nt. Also, that 'double shoulder' sharp peak is VERY characteristic of a successful library preparation with both miRNAs and piRNAs present, in my experience :)
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