RobertsLab
Proceeding with low-count sRNA-seq reads
Many of the timeseries P.evermanni samples have extremely low read counts (which is expected, based on Azenta sequencing report). Several also have very high counts, much higher than any of the A.pulchra samples. The quality metrics don't look dramatically different.
How should I proceed with these samples for miRNA discovery, using ShortStack? Should the low-count samples be excluded?
P.evermanni counts:
A.pulchra counts (for comparison):
P.evermanni trimmed MultiQC report A. pulchra trimmed MultiQC report
Time-series? - why is so varied across samples @kubu4 - any ideas ?
On Mon, May 12, 2025 at 5:26 PM Kathleen Durkin @.***> wrote:
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I'd review the Azenta QC and/or Azenta sequencing reports. I'm fairly certain Azenta was told to "proceed with best effort" for this project, so that suggests there were concerns prior to library prep.
Azenta QC stuff is likely in an issue somewhere.
In theory, the Azenta sequencing report (HTML file) should be in the corresponding nightingales folder on Owl.
I can try to track these down later today or tomorrow.
In meantime- Kathleen -no filter necessary for EPIMAR talk- will come into play for manuscript
On Tue, May 13, 2025 at 7:02 AM kubu4 @.***> wrote:
kubu4 left a comment (RobertsLab/resources#2187) https://urldefense.com/v3/__https://github.com/RobertsLab/resources/issues/2187*issuecomment-2876657329__;Iw!!K-Hz7m0Vt54!gxIKcfPneV9MBO3cY9NVvaOAmjiUIT5E5STp7wZphiEth5FhBUgrwvw93d31pFyWsbG6lumdLsjupw-qE-W49as$
I'd review the Azenta QC and/or Azenta sequencing reports. I'm fairly certain Azenta was told to "proceed with best effort" for this project, so that suggests there were concerns prior to library prep.
Azenta QC stuff is likely in an issue somewhere.
In theory, the Azenta sequencing report (HTML file) should be in the corresponding nightingales folder on Owl.
I can try to track these down later today or tomorrow.
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I think these results are not surprising. Many of the RNA samples provided to Azenta for this project (which split the submitted RNA between this and regular RNA-seq) fell below QC thresholds, including below the minimum input RNA required for each type of library prep.
I've attached Azenta's QC reports here, for cross referencing.
SampleQC_1-80_30-1047560508_a3dde5ed-6929-4b1b-9d96-f8807097ad1f.pdf SampleQC_81-117_30-1047560508.pdf Sample QC report of_30-1047560508_240828033818.pdf
Here's a link to the Azenta report for the raw data, which exhibits an extremely large range of read counts across all samples (it's interactive, so you can click on columns to sort the sheet):
https://owl.fish.washington.edu/nightingales/E5-coral-time-series/30-1069297013/Azenta_30-1069297013_Data_Report.html