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Dear Colleagues. I'm looking at small RNA fragments in my samples associated with or derived from mRNAs. I prefer to use the running window generator in SeqMonk because I can then annotate against various features without having to redefine the probes. The question is, what are the best settings to use? The libraries are typical NEBNext generated from 18-21 and 23-30 nt size fractionated RNA. I'm not normalising the read counts but I am rejecting duplicate reads. All advice greatly appreciated.
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Data variability is still an issue in sequencing technologies despite the advances in reproducibility and accuracy of these platforms. But the problem does not originate in the sequencing itself, but in the previous steps, before the sample reaches the sequencer.
The first step is collection, followed by preservation and sample preparation for analysis. Most scientists overlook those steps, but not being careful might just be skewing the experiment’s results.
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