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Old 11-04-2019, 08:30 PM   #1
Location: California

Join Date: Nov 2019
Posts: 20
Default Low data output from questionable sequencing facility

Hi all,

I have been getting some questionable runs from a sequencing facility and was wondering if there could be any clues as to whether it is their fault or my own fault in lab prep.

I am sequencing fish gut microbiome libraries on a Miseq 2x300 and the first run I sent off was great (was a different facility); lowest sample had 30k reads. Average was 100k per sample about.

Then we switched facilities and since then I have never achieved anything close to my first run. And these are all very similar libraries, with near identical protocols except now we use MagBind bead cleans instead of AMPure (very similar product). Now all my runs average about 30k reads per sample, many getting less than 5k, and my latest Miseq run only generated 7 million reads total . I've attached a picture of QC reports for the good run and an example of a current bad one.

Is it that easy for a facility to mess up so many miseq runs? They have been loading at 12-13pM with 20-25% PhiX. My last library was qubited at 26nM.
Another piece of info is that the core recently dropped their prices in half. Don't want to give specifics but it is less than $800. So VERY cheap IMO. Not sure if this could influence what goes on at a facility.

From the reports %PF is around 93-95% and the Q30% is around 84-89% for these runs.

Let me know if I should provide any other information.
Any feedback is much appreciated!
Attached Images
File Type: png Run comparison.png (71.8 KB, 14 views)

Last edited by samd; 11-06-2019 at 06:46 PM.
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