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Old 12-17-2012, 04:53 AM   #1
pmiguel
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Thumbs up MiSeq best result?

I am calling out all who think they have good MiSeq runs. Gaze at the perfection of this run and weep at the inadequacy of your wretched instruments:



But seriously...
Accidently clustered at a higher density than normal. But the results were still good. Raw cluster density was 1154 Kclusters/mm^2. 8.8 billion bases total. 6.8 billion bases >Q30.



Kind of interesting, this indicates that 0.6x0.4 = 24% of read pairs have zero sequencing errors in them!


And, just to add historical perspective, this is around the amount of data generated by 13 GS-FLX runs. (And we would bill out a GS-FLX run at 5x the cost of a MiSeq run.) Also, at 80 kb per 3730XL run, we would need 13,000 of them to equal the output of this single MiSeq run. That is 3 years of non-stop runs on a 3730XL!

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Old 12-17-2012, 08:21 AM   #2
GenoMax
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That is .. normal genomic DNA, I assume.

How about running some 16S samples.
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Old 12-17-2012, 11:59 AM   #3
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That is .. normal genomic DNA, I assume.

How about running some 16S samples. :)
No, an RNA seq library.

We will be doing some 16S fairly soon.

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Old 12-18-2012, 06:15 AM   #4
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I mentioned in other thread that I was getting decent results at density of as much as 1500. My problems is associated more with short libraries made from ancient DNA, so pushing longer than 2x151 cycles makes little gains. But with density of 1100 I got about 75% of >30Q with 2x151 cycles.
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Old 12-18-2012, 08:58 AM   #5
Jean
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How much PhiX spike-in?
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Old 12-18-2012, 09:19 AM   #6
yaximik
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If this is question to me, I do not spike-in as I am using only fastqc framework - genomes are too big to be processed on-board
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Old 12-18-2012, 09:49 AM   #7
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No phiX on our (Purdue's) end. We ended up with about 0.1% of the reads which potentially map to phiX but then we always seem to get this low-level matching. As for the overall stats:

33,676,040 raw reads
8,452,686,040 raw bases
251 length

33,156,566 reads after adapter and quality trimming
7,559,421,635 bases after trimming
30-251 length with a mean of 227
1% of the sequences lost to trimming
10% of the bases lost to trimming

It is an incredible amount of data, at least for those of us who have been around for a long time. Equivalent to 2x human genome coverage for, what, $3000 or so.



Overall very comparable to other miSeq runs we have done.
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Old 12-19-2012, 03:39 AM   #8
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Rick,
What? We did spike in phiX!
We ostensibly spiked in 1% phiX -- but it turned out that only 0.3% of the reads derived from phiX.
phiX gives the % perfect read plot.

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Old 12-19-2012, 03:48 AM   #9
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We have not had any problems getting the clusters to register without spiking in phiX with 16S runs. It is the quality values assigned to the bases (in latter half of 2 x 250 bp runs), where the problem occurs. Sequences being generated have been acceptable for the end users with no problems about the actual base calls.

Would be curious to see how you fare with your 16S runs. I assume you will be spiking in phiX.
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Old 12-19-2012, 04:47 AM   #10
westerman
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In reply to Phillip:

Ooops. Don't know what I was thinking. Obviously I wasn't. :-( Should have looked at the lab notebook before spouting off to the world.
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Old 12-19-2012, 11:33 AM   #11
pmiguel
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Quote:
Originally Posted by GenoMax View Post
We have not had any problems getting the clusters to register without spiking in phiX with 16S runs. It is the quality values assigned to the bases (in latter half of 2 x 250 bp runs), where the problem occurs. Sequences being generated have been acceptable for the end users with no problems about the actual base calls.

Would be curious to see how you fare with your 16S runs. I assume you will be spiking in phiX.
We will likely spike in genomics libraries of some sort. That way some useful data is generated from the genomic 'ballast' library.

So you are saying that the quality values assigned are inaccurate (lower than they should be)?

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Old 12-19-2012, 06:07 PM   #12
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We should be getting back our MiSeq 16s results within the next day or two- did a 50% spike-in of gDNA under the recommendation of the center. Right now our SOP is to find some genome in our lab that needs sequencing and use that to up the diversity on the run. Otherwise it's 90 barcoded samples...I'll let you guys know how it went when we get it!
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Old 12-20-2012, 04:04 AM   #13
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Quote:
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We will likely spike in genomics libraries of some sort. That way some useful data is generated from the genomic 'ballast' library.

So you are saying that the quality values assigned are inaccurate (lower than they should be)?

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That is the assumption we are going on. We (meaning the people in the wet lab) are using custom primers, so genomic "ballast" library is not an easy option.
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Old 12-20-2012, 10:10 AM   #14
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Default Our record

Was the incredible amount of data you got from one run using an upgraded MiSeq? We have not had the upgrade yet. Our record with RNAseq libraries has been 2.5 Gb of calls >Q30 (1781 K/mm2, 78.5% pass, 13.37 M raw reads, 2x150bp). RNAseq libraries allow higher resolvable cluster density than Nextera yeast genome libraries. Our record with Nextera libraries is 2.4 Gb of calls >Q30 (1262 K/mm2, 90.1% pass, 9.98 M raw reads, 2x150bp).

We suspect that the higher cluster density with RNAseq libs is due to there being fewer longer fragments in the library, which would presumably form larger clusters that would be harder to resolve. We would love to know about tricks to achieve higher cluster densities with Nextera libraries. We already modified the PCR in the protocol by reducing the extension time from 3 min to 1 min and adding two more cycles (7 total).
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Old 12-20-2012, 10:35 AM   #15
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Originally Posted by creeves View Post
Was the incredible amount of data you got from one run using an upgraded MiSeq?
Yes.
Quote:
Originally Posted by creeves View Post
We suspect that the higher cluster density with RNAseq libs is due to there being fewer longer fragments in the library, which would presumably form larger clusters that would be harder to resolve. We would love to know about tricks to achieve higher cluster densities with Nextera libraries. We already modified the PCR in the protocol by reducing the extension time from 3 min to 1 min and adding two more cycles (7 total).
You want to remove the larger fragments, you mean? You could do an Ampure upper cut, right?

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Old 01-17-2013, 08:06 AM   #16
lorendarith
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Quote:
Originally Posted by westerman View Post
33,676,040 raw reads
8,452,686,040 raw bases
251 length

33,156,566 reads after adapter and quality trimming
7,559,421,635 bases after trimming
30-251 length with a mean of 227
1% of the sequences lost to trimming
10% of the bases lost to trimming
What was the quality trimming exactly?
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Old 03-05-2013, 03:31 AM   #17
matth431
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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 at 03:44 AM.
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Old 01-06-2017, 07:42 AM   #18
Shimul
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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,

Shimul
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Old 01-06-2017, 08:08 AM   #19
pmiguel
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Originally Posted by Shimul View Post
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,

Shimul
Sure, those metrics would consistent with the start of a good run.

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Old 01-06-2017, 09:09 PM   #20
Shimul
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Quote:
Originally Posted by pmiguel View Post
Sure, those metrics would consistent with the start of a good run.

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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?

Many thanks.

Shimul
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250 base reads, 500 cycle kit, cluster density, miseq, v2 hardware

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