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Originally posted by westerman
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What was the quality trimming exactly? -
Just got 10.3Gbp raw data, histogram suggested 7.0Gbp >Q30 by end of 251|6|251 run. Useable sequence (post-QC trim) checking it on Genomics Workbench probably closer to 9Gbp. About 39.5M reads with average length of 225bp.
Cluster density of 1300K/mm2 - same library gave ~3.5Gbp on a v1 flowcell with 2x151bp reads (clustered at 8pM).Last edited by matth431; 03-05-2013, 04:44 AM.Comment
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Hi All,
I am finally running of my bacterial pooled DNA library (which I made using Nextera XT) on the Miseq using MiSeq V3. I have checked the machine now and it shows that after 70 cycles, the >=Q30 is 96%, cluster passing filter 91.4%, and the cluster density is 1092K/mm.
Do you think that the run is going well?
Hope to hear back from you soon again!
Cheers,
ShimulComment
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Sure, those metrics would consistent with the start of a good run.Originally posted by Shimul View Post
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PhillipComment
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The Q30 is now 83.1% after 250 cycles, yesterday it was 96% after 70 cycles. But rest of the parameters are consistent: clusters PF 91.4%, cluster density 1092K/mm^2, which are the same as yesterday. Do you speculate anything? DO you think that the run will be okay?Originally posted by pmiguel View Post
Many thanks.
ShimulComment
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Sounds like this is your first ever run and you are anxiousOriginally posted by Shimul View Post
Cluster density does not change over the run duration. As @nucacidhunter pointed out Q scores may keep dropping. Enjoy the weekend and don't worry about the run.Last edited by GenoMax; 01-09-2017, 08:51 AM.Comment
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A correction Geno, %PF does not change. PF clusters are set at cycle 25. Q scores of course are calculsted on each cycle and later cycles always have lower quality than earlier ones.Originally posted by GenoMax View PostSounds like this is your first ever run and you are anxious
Cluster density does not change over the run duration. As @nucacidhunter pointed out the PF% will likely continue dropping as will the Q scores. Enjoy the weekend and don't worry about the run.
Shimul, relax, your run is going fine.Comment
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I think it is less accurate quote of my post (#65) from the following:Originally posted by GenoMax View PostSounds like this is your first ever run and you are anxious
Cluster density does not change over the run duration. As @nucacidhunter pointed out the PF% will likely continue dropping as will the Q scores. Enjoy the weekend and don't worry about the run.
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@nucacidhunter: Please accept my apologies. Thank you @kmcarr also. I should have checked my post more carefully (I have made necessary correction above).Originally posted by nucacidhunter View PostComment
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
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I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...
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