Entering edit mode
15 months ago
sithlordashley
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0
Hello,
My lab isolated miRNA from samples and outsourced the quality control (Nanodrop, TapeStation) as well as Illumina sequencing and analysis. The outcome was very low miRNA transcript counts. I am trying to interpret a FastQC to see if the outsourced analysis could be improved by trimming around the 29bp mark. I am used to seeing quality decreasing over time, but not dipping sharply and returning to high quality score. Furthermore, there are other issues such as per sequence base content ranging from ~ 6 - 70% at times,
My goal is to improve the counts and improve quality of analysis, if possible. If the reads were trimmed at 29bp, would that be too low?
Find out which kit was used for this prep. Follow the kit instructions to find and remove a kit specific miRNA adapter (more than likely be the case) and then you should be left with short reads < 30 bp that you can align with
bowtie v.1.x(ungapped alignments) or use a standard miRNA analysis pipeline.Note: Upload images of your fastQC if you can. Hard to imaging what the data looks like based on text description.
Thank you for your reply! My apologies for forgetting the attachment, the FastQC report is now attached to the original post. We outsourced library prep so I will check the specific miRNA adapter from the kit they listed in their methods. I am primarily looking for accurate critique that I could forward back to the university that performed the work because we are unable to work with the generated data.
Once you properly trim the data you will hopefully find that things will look much better. There is definitely signal there in first ~25 bp which is consistent with miRNA data.