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Old 11-06-2019, 01:36 PM   #13
itstrieu
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Location: Atlanta

Join Date: Nov 2018
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Quote:
Originally Posted by samd View Post
Hi SNPsaurus,

Ok so after discussing with them the 11M reads simply referred to the forward reads. The 23M PF refers to the forward and reverse. I see a 40% undetermined reads metric. Which is a bit high compared to 25% on my last run so I wonder if this is an indexing issue vs a sequencing issue?

@itstrieu: Damn I wish I could get those numbers. Would you recommend upping my concentration to 17pM instead of 13? Or is that dependent on other factors.
I would say it is library dependent and what metrics you are aiming for. For V3V4 sequencing, we use spacer primers to add diversity to the run.

For V4 sequencing, we use 515F (Parada)–806R (Apprill) primers from EMB with a V2 500 cycle kit and we usually get about 35M PE reads that pass filter. For this library and kit, we load at around 8.75 pM but keep a log to have a floating average to determine the loading concentration for the next run.

Also it might depend on the MiSeq because we have two MiSeq and one performs slightly better. I usually vary the concentration in increments of 0.25 0.50 pM to be careful not to over cluster.

Last edited by itstrieu; 11-06-2019 at 01:42 PM.
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