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Old 11-24-2016, 11:53 AM   #101
Brian Bushnell
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If the cost is reasonably similar, I'd go with HiSeq 2000. But perhaps you can get a sample of NextSeq data for your project, on a library you already ran on the HiSeq, to compare without committing yourself?
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Old 11-24-2016, 05:45 PM   #102
Michal2213
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Default NextSeq suitable for allele-specific analysis?

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Originally Posted by Brian Bushnell View Post
If the cost is reasonably similar, I'd go with HiSeq 2000. But perhaps you can get a sample of NextSeq data for your project, on a library you already ran on the HiSeq, to compare without committing yourself?
Thank you for reply! I would do the same but with NextSeq we are getting data within a week as we have direct access and with HiSeq2000 in the sequencing core one need to wait in many cases more than a month. I guess we will need to go for sequencing the same library on both libraries as you suggested as well.
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Old 02-27-2017, 08:57 AM   #103
dsobral
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Just to add our 2 cents.

We have a MiSeq running since 2013, and after some hickups we're now stable with it and reasonably happy.

We just recently installed a NextSeq500 and our first tests are not great. Q30 is >80%, but there are many low quality bases (constantly Q=14 "/"), and the worst part is that instead of being towards the end, they seem a bit randomly distributed. When comparing PhiX in a 2x150bp NextSeq with a 2x250bp MiSeq, after alignment I see a 0.2-0.3% error rate with MiSeq and 0.9-1% error rate with NextSeq (1M sampled reads). In the "randomly" distributed Q=14 bases I seem to notice more A to T transitions, but I didn't have time to gather more systematic statistics... If I do quality trim on the MiSeq I can easily get higher quality data, with the NextSeq since its randomly distributed is harder...

We've complained to the Illumina people, let's see what they say...
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Old 02-27-2017, 11:25 AM   #104
Brian Bushnell
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We've complained to the Illumina people, let's see what they say...
I don't believe that Illumina officially admits that NextSeq is lower quality than HiSeq 2500 or MiSeq. Error rate is not part of the platform specification, just quality scores.
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Old 03-09-2017, 11:41 PM   #105
alexhaj
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Good work Brian! I am skeptical about your claim that NextSeq has less crossover than HiSeq or Miseq however. Would you please provide some data to back this up. And if that is the case maybe it's simply because demultiplexing is being done by CASAVA v2 on NextSeq and CASAVA v1 on HiSeq and Miseq. What if you did demultiplexing yourself, taking into account Quality scores (which I assume is not typically done).
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Old 03-13-2017, 02:39 PM   #106
Brian Bushnell
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Good work Brian! I am skeptical about your claim that NextSeq has less crossover than HiSeq or Miseq however.
We did fairly extensive analysis on this, and the results were never strongly reproducible from one run to the next on a given platform, but this one result (NextSeq outperforming HiSeq/MiSeq in crosstalk) was very consistent. Bearing in mind that we only had a single NextSeq machine at the time.

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Would you please provide some data to back this up.
I may try to dig it up if I have some time; there is no comprehensive single report with all of it so it would be a lot of work.

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And if that is the case maybe it's simply because demultiplexing is being done by CASAVA v2 on NextSeq and CASAVA v1 on HiSeq and Miseq. What if you did demultiplexing yourself, taking into account Quality scores (which I assume is not typically done).
I did do the demultiplexing manually, which is why (for example) mergebarcodes.sh and filterbarcodes.sh are in the BBTools package; I wrote them just for this experiment Even aggressive filtering on barcode quality was unable to substantially impact the relative differences between the platforms.
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Old 10-29-2017, 11:27 PM   #107
ymc
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Unfortunately, Illumina's taken a turn for the worse again. I just analyzed some recent data from the NextSeq, HiSeq2500, and HiSeq 1T platforms of the same library. The NextSeq data is dramatically worse than last time I looked at it. Error rates are several times higher, there's a major A/T base frequency divergence in read 2, and the quality scores are inflated again at ~6 points higher than the actual quality. More disturbingly, the HiSeq quality scores are completely inaccurate now, as well, though the actual measured quality is still very high - average Q33 for read 1 and Q29 for read 2 for HiSeq2500, versus Q24 for read 1 and Q18 for read 2 on the NextSeq (those numbers are as measured by counting the match/mismatch rates from mapping, so essentially, NextSeq has roughly 10X the error rate of HiSeq). But the measured discrepancy between claimed and measured quality scores for the HiSeq2500 and HiSeq 1T are BOTH worse than the NextSeq, despite the NextSeq having binned quality scores, and as you can see there are large regions of quality scores simply missing from the HiSeq2500, such as Q3-Q11, Q17-Q21, and Q29. There are clearly major problems with Illumina's current base-calling software, as quality score assignment has drastically regressed since last time I measured it.

You can see the graphs in this Excel sheet that I've linked. "Raw" is the raw data, "Recal" is after recalibration (which changes the quality scores but nothing else). "NS" is NextSeq, "2500" is HiSeq2500, and "1T" is HiSeq 1T which unfortunately was only run at 2x101bp instead of 2x151bp on the other 2 platforms.

https://drive.google.com/file/d/0B3l...ew?usp=sharing
Are there any updates for the current state of nextseq? Is it still this bad or return to the good quality when v2 came out?
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