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Old 01-30-2017, 07:59 AM   #1
jhi_pete
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Default MiSeq v3 600 bp improvements?

I have heard a rumour that the long standing MiSeq v3 600 bp kit chemistry problems which led to poor quality read 2 data have recently been solved by Illumina. Anyone know if this is true?
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Old 01-30-2017, 08:34 AM   #2
nucacidhunter
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It is true and for high diversity libraries output is according to specification. Amplicon sequence quality also has improved but as before cluster density should be reduced and spiked in with PhiX.
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Old 01-30-2017, 10:55 AM   #3
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I just started my first 600-cycle run in over a year so hopefully it's true! I'll try to update in a couple of days when it's finished.
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Old 01-31-2017, 08:31 AM   #4
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Can anyone confirm this? I need to do some 2x300 sequencing for Ig RNA-seq experiments. Would be awesome if I could use the MiSeq.
Thanks,
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Old 01-31-2017, 08:32 AM   #5
Markiyan
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Thumbs up Yes, the last few 2x300 runs were way better than 1 year ago.

Yes, the last few runs were way better (touch the wood).

If doing amplicons, please use a lot of phiX or shotgun library (up to 20%) (esp. if fist 25 bp's are the same). It would not hurt, and gains in data quality/yield from better dephasing calculation far outweigh the losses from 5%-20% phiX sequence spike in.

If doing cDNA amplicon analysis: make sure you do not use oligoC oligos in your amplicons, because the read quality would go through the floor if there are 10-14C's or G's in a row, and you may also get non-specific amplification of rRNA (rcDNA), reducing usable data yield by 1-2 orders of magnitude.

Also lower the loading density for 500-600bp amplicons, if you need high quality reads for your analysis.

Obviously do not forget, that the longer the amplicon or the read is, the more sensitive it becomes to the DNA sequence content.
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Old 01-31-2017, 06:18 PM   #6
Brian Bushnell
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Quote:
Originally Posted by plyguy69 View Post
Can anyone confirm this? I need to do some 2x300 sequencing for Ig RNA-seq experiments. Would be awesome if I could use the MiSeq.
Are there alternatives other than Sanger? Or, what was your backup plan?

@Markiyan - thanks for the advice, I'll pass it on!
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Old 02-01-2017, 01:20 AM   #7
jhi_pete
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Thanks to everyone for their positive replies, that is very good news. We have ordered up a 600 bp kit and will test it ourselves soon and let you know how we get on.
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Old 02-01-2017, 02:09 AM   #8
Markiyan
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Default 550bp+ full amplicon sequencing platforms...

Quote:
Originally Posted by Brian Bushnell View Post
Are there alternatives other than Sanger? Or, what was your backup plan?
1. Pacbio RSII CCS - should work quite nicely for amplicons up to 2-4kb.

2. If you can cope with 2-4% systematic errors - Oxford Nanopore 2D.

3. There used to be Roche's systems - FLX+ and Junior, but the reagents for them are no longer available... :-(
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Old 02-21-2017, 05:49 AM   #9
jhi_pete
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Quote:
Originally Posted by microgirl123 View Post
I just started my first 600-cycle run in over a year so hopefully it's true! I'll try to update in a couple of days when it's finished.
Hi Microgirl. How was your 600 cycle run?
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Old 02-21-2017, 05:53 AM   #10
microgirl123
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Not good

It was an amplicon run, but the cluster density was good (~600K) with 20% phiX, so I expected the Q30 to be much better than 64% overall. It was only 54% for Read 2. Both reads only got out to about 150 bp before dropping off dramatically. The investigator decided it was definitely not worth trying to use the 600-cycle kit again!
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Old 02-21-2017, 05:55 AM   #11
jhi_pete
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Quote:
Originally Posted by microgirl123 View Post
Not good

It was an amplicon run, but the cluster density was good (~600K) with 20% phiX, so I expected the Q30 to be much better than 64% overall. It was only 54% for Read 2. Both reads only got out to about 150 bp before dropping off dramatically. The investigator decided it was definitely not worth trying to use the 600-cycle kit again!
Oh dear! That's unfortunate, sorry to hear that.

Anyone else with recent experience running the 'new' 600 bp kits? We plan to use one this week or next....

Last edited by jhi_pete; 02-21-2017 at 05:56 AM. Reason: More info
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Old 02-21-2017, 05:59 AM   #12
GenoMax
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Quote:
Originally Posted by microgirl123 View Post
Not good

It was an amplicon run, but the cluster density was good (~600K) with 20% phiX, so I expected the Q30 to be much better than 64% overall. It was only 54% for Read 2. Both reads only got out to about 150 bp before dropping off dramatically. The investigator decided it was definitely not worth trying to use the 600-cycle kit again!
Am curious about the focus on the Q-scores. Is the investigator looking to call SNPs? Aligning to reference sequence should be no problem irrespective what the Q-score is.
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Old 02-21-2017, 07:39 AM   #13
Markiyan
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Lightbulb Adapter dimers?

Quote:
Originally Posted by microgirl123 View Post
Not good

It was an amplicon run, but the cluster density was good (~600K) with 20% phiX, so I expected the Q30 to be much better than 64% overall. It was only 54% for Read 2. Both reads only got out to about 150 bp before dropping off dramatically.
This looks like issues with adapter dimers to me - what was yours beads ampure cleanup ratios, and any slight bump up on the agilent trace in the region of 100-200bp?

Basically significant amount of the clusters on the run contained shorter templates, and once the end of these is reached (140-160 bp), it wrecks RTA dephasing calculations.

since templates in the 150-200 bp cluster very efficiently compared to 500-800 bp ones, it takes only 3-5% of the shorter contaminant to wreck Q scores in the run.

To minimize it avoid repetitive freeze thaw cycles of the Illumina adapters (they can lose T tails and self ligate) and make sure no nucleases contamination is present during the ligation.
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Old 02-21-2017, 10:29 AM   #14
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Quote:
Originally Posted by microgirl123 View Post
Not good

It was an amplicon run, but the cluster density was good (~600K) with 20% phiX, so I expected the Q30 to be much better than 64% overall. It was only 54% for Read 2. Both reads only got out to about 150 bp before dropping off dramatically. The investigator decided it was definitely not worth trying to use the 600-cycle kit again!
Tech support has told me not to cluster below ~750k even for v2 even for amplicon runs. If it's 16S, I had a particular set of samples from some sort of bird guts that I basically can't run by themselves even with 20% phiX because R2 is so bad. There seems to be something happening between cycles 10-30 of R2 that is actually sequence dependent. The way my facility is set up, I mix and match small projects on runs so this is addressable by splitting up the bird samples across many runs.

The suggestion that there may be a primer diamer issue is reasonable, but you also may need to up your density a bit.
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Old 02-21-2017, 06:36 PM   #15
nucacidhunter
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Quote:
Originally Posted by microgirl123 View Post
Not good

It was an amplicon run, but the cluster density was good (~600K) with 20% phiX, so I expected the Q30 to be much better than 64% overall. It was only 54% for Read 2. Both reads only got out to about 150 bp before dropping off dramatically. The investigator decided it was definitely not worth trying to use the 600-cycle kit again!
In addition to above comments it could be a bad batch as well. My current runs Q30 are above %70 specified by Illumina for 2x300 reads:
Attached Files
File Type: pdf MiSeq.stats.pdf (92.4 KB, 24 views)
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Old 02-22-2017, 08:03 AM   #16
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We did a 2x300 run for a TCR library recently. Passed spec (77.5% Q30 @ 939k with 15% PhiX), but clear tailing towards the ends of both R1 and R2 as well.

I could see error rates approaching 5% being problematic for many if not most applications.

We'll be sticking with 2x250 or 2x150 chemistry for the time being, unless specifically requested by a client.
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File Type: pdf median_Q30.pdf (49.9 KB, 25 views)
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Old 02-23-2017, 02:43 PM   #17
nucacidhunter
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To my knowledge error rate is not included in Illumina specifications. Q scores drop after cycle 250 is a known characteristic of the chemistry and for some applications it will not be detrimental.
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Old 03-23-2017, 02:50 AM   #18
Bukowski
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So.. no this issue is not resolved. I have an email from Illumina today which I will summarise.

The issue is that there is a byproduct in their manufacturing process of fluorescent nucleotides. This byproduct results in NON-CLEAVABLE NTPs - so they limit strand growth. Obviously this has limited effects on the number of clusters per cycle, but does become more acute on 600 cycle kits as the problem 'builds'. You get a lower cluster density, and lower Q30 and higher error rates on the later cycles.

Illumina can now *detect* this byproduct and know what levels impact performance, which sounds to me like there might be batch to batch variation, and have put in place measures to screen for it, therefore they are still expecting improvements 'in the near future' as newer material hits the manufacture pipeline. I am also told there will be a new announcement soon, with new timelines for resolution.
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Old 03-23-2017, 06:27 AM   #19
plyguy69
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Thumbs up thanks for the update

I've have also heard that the problem wasn't resolved, but never had details as to the cause.

Thanks!
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Old 03-23-2017, 06:47 AM   #20
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Many thanks Bukowski, that's the first logical explanation I have heard. Looking forward to any announcements from Illumina going forward.
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