MiSeq/cluster density problems

[francesca3877]

Hi everyone,
my name is Francesca. Yesterday, I completed a new MiSeq run but I had some problems. I use target sequencing technique by Haloplex and I loaded 12 samples. For quantification, I use Tape Station and the results were good. But, the results of MiSeq run was opposite in fact, I obtained a good cluster density 1,337k/mm2 but, the quality was to low 6,1%. I don't understand because I load two of these sempales in other experiments and the results are good.

[bunce]

Hi francesca3877,

There are many, many reason for a poor sequencing run - the #1 cause is usually clustering density (are using using v2 or V3 chemistry). You can't just look at the clustering number and believe it - when a run is over clustered the density maxes out and gives you erroneous numbers.

have a look at this illumina help guide - and check out the thumbnail images on your run.

https://support.illumina.com/content/dam/illumina-marketing/documents/products/other/miseq-overclustering-primer-770-2014-038.pdf

[GenoMax]

Hi everyone,
my name is Francesca. Yesterday, I completed a new MiSeq run but I had some problems. I use target sequencing technique by Haloplex and I loaded 12 samples. For quantification, I use Tape Station and the results were good. But, the results of MiSeq run was opposite in fact, I obtained a good cluster density 1,337k/mm2 but, the quality was to low 6,1%. I don't understand because I load two of these sempales in other experiments and the results are good.

Your best bet is to open a ticket with Illumina tech support for any run that does not meet published spec. Tech support can look at the metrics of the run and determine if there is an issue. If there is an issue you would get free replacement reagents, otherwise you will get an explanation of why you are seeing the results you have.