[samd]

Hi SNPsaurus,

Interesting. I have heard of the spacer primers but I think I am past the point in my PhD of redoing everything. But it would be nice.

This might be a dumb question but I thought a Miseq spits out 25M but I am seeing you and others say 35M or 50M? How is this possible?

[itstrieu]

You can increase cluster density by increasing the loading concentration.

In my post they are paired end reads so double that if you where doing single end reads. Here is a link how many reads you should expect for various MiSeq kits but it is dependent on cluster density. https://www.illumina.com/systems/seq...fications.html

Edit. Sometimes you hear the word cluster and reads used together. I believe that for a v3 kit, it can generate around 25M unique cluster and each cluster can do two reads for paired end so it would output 50 M reads

[samd]

Ok so I am guessing the "25M total reads" on Basespace actually means 50M since I did PE. Thanks for the suggestion I will look into that.

One thing I just remembered is the QC results were quite different from the first "good run" and the subsequent "bad runs". The good run has a nice skinny peak and the bad runs have lumpy peaks which I guess would be attributed to non-specific binding of primers? I've attached to the post in case anyone is interested or has any insight into that.
Again, thanks for all the feedback!
Sam

[itstrieu]

Ok so I am guessing the "25M total reads" on Basespace actually means 50M since I did PE. Thanks for the suggestion I will look into that.

One thing I just remembered is the QC results were quite different from the first "good run" and the subsequent "bad runs". The good run has a nice skinny peak and the bad runs have lumpy peaks which I guess would be attributed to non-specific binding of primers? I've attached to the post in case anyone is interested or has any insight into that.
Again, thanks for all the feedback!
Sam

If the total reads is 25M under the indexing QC tabs in BaseSpace, it is actually the total PE reads. Under the Metrics tab, READS PF will be half of that. I would ask if they could rerun the library but at a higher concentration to target for a cluster density of around 900 K/mm2 for more reads.

[samd]

Hi itstrieu,

I see. Well I guess I am getting very low outputs then.. I will run that suggestion by them. It is just strange because my first run at Berkeley which I consider "good" was done at 12pM and I even tried upping it to 13pM here at UCLA and ended up getting fewer reads.

[ddabiri]

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?

In relation to this observation, I recently started a run that ended with a Qscore of 78% and a Read PF of 96%. It had a total read of 4.9M. Its underclustered as a result of using the Qubit value for loading rather than using the qPCR value and also we multiplexed 350 samples (amplicon). However, one surprising event is that half of the samples are little or no reads while the other indexed samples have the 4.9m reads generated. Any explanation for this.

Thanks