Low data output from questionable sequencing facility

[GenoMax]

2. Cluster density was about 477 if I remember correctly

That does not jive with read numbers. Are you referring to 23M total reads (R1+R2) or passing clusters?

That cluster density if not very high to get to 20+M clusters.

[samd]

Hi all,
appreciate the responses

@nucacidhunter
1. Primers are the EMP 515-806 V4 region
2. Cluster density was about 477 if I remember correctly
3. Just 1 library. Usually I was doing 160 samples per run and this time I reduced the run to about 100 samples and still got crummy results
4. Read output was actually 25M and then 23M PF which I guess is great actually but then 40% were undetermined and then when I import the fastq files into QIIME2 I only get about 7 million reads (this is before dada2, so still the entire files).

[SNPsaurus]

The $800 is very very low. Even twice that would be on the low end at many service providers for 2x300 v3 MiSeq.

Have you compared Qubit numbers to qPCR to see if there is a mismatch in those approaches to quantifying your library?

[luc]

This is using custom sequencing primers?

The heating and cooling elements are not calibrated exactly the same for all MiSeqs (by Illumina). Some risky custom sequencing primer designs can work with some Miseqs better than others.

[nucacidhunter]

More info for following would be useful:
1- Are the libraries 6S V regions and overall prep workflow
2- cluster density
3- How many libraries are multiplexed
4- Read output

[samd]

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!
Thanks,
Sam