We are experiencing problems with MiSeq runs (using 600-cycle V3 kits) at our core facility: Two week ago they ran a PhiX standard and a customer-prepared library- both worked fine. However, when they ran another library (from source A) and got no clusters. After that they rebooted the instrument and performed another volume test, which passed. Then they ran our sample. Again, the clusters had very low intensity and none of them passed filter. The clusters just didn't look right to them. I am attaching the RunInfo and RunParameter files here. We would appreciate any inputs.
-
-
Here is the message from Illumina:
"I can't say for sure if the run is over-clustered, but it does look like it is low diversity and perhaps a lack of PHiX caused it to loose focus. Depending on what the library is, I would recommend double checking the primers and the PhiX spike in". -
Recommendation from Illumina is clear and your facility needs to follow it. Spike phiX in the next run, if that was not done (it sounds like it was not). That is the only way to allow runs with low nucleotide diversity to go through.Comment
-
What kind of libraries are they?
We had a non-standard library from a customer a few months ago that would kill the whole flowcell, PhiX and all. I discussed it here. Illumina speculated that there might have been some PCR primer that didn't get cleaned out that was preventing the libraries from annealing. A bead cleanup of the library eventually sorted it out.Comment
-
PhiX was added at 30%. Is there a way to tell whether the PhiX added was bad (degraded)?Originally posted by GenoMax View PostComment
-
The library is the mixtures of 16S and ITS amplicons. We prepared the library following The Schloss Lab Protocol and we had no problem for previous runs -even at 5% PhiX.Originally posted by GW_OK View PostComment
-
I'd try doing a bead cleanup and reloading.Comment
-
No problem with previous runs in 2013 and 2014, but could not get high quality sequences since 2015.Comment
-
What were the cluster densities like, when you did get clusters to form? What was the pass filter?Comment
-
On another poor run at end of 2015: cluster density (775 +/- 49 k/mm2) with 81.8% PF. Summary graphs attached.Comment
-
@phyllosphere: Have you seen this thread for 2x300 runs (http://seqanswers.com/forums/showthread.php?t=59558) on MiSeq. Not directly related to your original post but something to keep in mind. Great 2x300 runs with low diversity libraries are not going to happen with V3.Comment
-
Thanks @GenoMax. Yes I read almost all the threads related to MiSeq here (desperate to move on with our microbiome projects). We did get four separate good 2x300 runs with low diversity libraries on the same MiSeq (when it was relatively new), but are hitting the wall now despite repeated efforts with higher PhiX. I suspect the poor Illumina kits manufactured after 2015, as well as some of the aged components of our MiSeq, all contributed to the problem.Comment
-
I'd say this looks about par for the course for a v3 600 cycle kit, especially with low diversity.Originally posted by phyllosphere View PostComment
-
2x300 is longer than you need for the v4 16s and probably longer than you need for ITS depending on which primers you are using.Originally posted by phyllosphere View Post
Was your phiX freshly denatured?
If you rerun, do a 2x250, 2x300 just don't work well.
ETA-wrote that before I saw you second run metrics. That looks great for an amplicon run on V3 (which isn't a really high bar, but I wouldn't be thinking about rerunning that). Have you clustered the data and it's bad or are you thinking it's bad just based on the q30 and PF%Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.Comment
-
NGS has revolutionized clinical microbiology by offering new ways to identify and surveil pathogens without prior knowledge of the target. One popular approach is 16S rRNA and ITS amplicon sequencing, which targets specific microbial markers. But studies are increasingly incorporating shotgun metagenomics, a technique that sequences all DNA from a sample without targeting specific microbial markers. This approach allows more detailed taxonomic classification, detection...Today, 06:46 AM -
Tumors contain complex mixtures of cell populations that evolve in response to treatment and host-specific factors. Tissue biopsies provide only static snapshots of a tumor’s state. But what if we could follow that evolution over time using a minimally invasive technique?
Liquid biopsies can detect tumor-derived material circulating in the bloodstream, providing a way to study treatment response, detect emerging resistance, and discover new biomarkers. Circulating...10-14-2026, 09:55 AM
Comment