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This is an interesting thread - we also had a drop in MiSeq performance (in low complexity libraries) after upgrading to 2.6 and like some of you rolled back to 2.5. I was told there was nothing in the upgrade that might cause this but our runs turned to custard about the time we upgraded. I am staying put in 2.5 for as long as possible.
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Hi GenoMax, not in so many words, but they had reports of poor performance and the primary improvements in MCS 2.6 were for the Trusight Tumor 15 workflow, so rolling back was suggested if we weren't running that.Comment
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Just in case you haven't seen it: The 2.6 version is currently not available due to a "review"
From: https://support.illumina.com/downloa...s_updater.htmlThe recently-released MiSeq Updater v2.6.1.4 has been temporarily removed from our website for review. We will update customers as information becomes available.Comment
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Well it's clearly library specific, though I can't figure out how. I had a successful run last week 75% 7pM 16s amplicons, 20% 8pM truseq genome, 5% phiX. Tried running the problem library again-60% 6pM 16s amplicon, 10% ITS amplicon, 15% 8pM nextera genome, 15% phiX. Again the first read and the indices look great (700k, 86%PF, 16% aligned to phiX). Second read is horrible and only 3% aligned to phiX. This run is higher diversity (though still very low diversity by general Illumina guidelines) and lower clustering than the last successful run.Last edited by thermophile; 02-10-2016, 02:19 PM.Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.Comment
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This run finally worked on a different machine. FAE is coming out next week to check the temps on both machines and to adjust my machine to match the one that worked.Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.Comment
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@Number6: We finally hit the "lost focus" error late in cycle on one of runs and were advised by tech support to roll back to MCS v.2.5.x. So far so good.Comment
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My temp control module is bad!! It would hit a temp but then not hold it. FAE is here replacing itMicrobial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.Comment
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Yet another run failed (after the TCM was replaced). Second level support suggested adding more primers since the TM of the primers (v4 bacteria, aka the ones that most microbiome people use) is low 62-66. This seems to have fixed the issue. I'll see how long this fix lastsMicrobial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.Comment
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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...06-18-2026, 07:11 AM -
Data variability is still an issue in sequencing technologies despite the advances in reproducibility and accuracy of these platforms. But the problem does not originate in the sequencing itself, but in the previous steps, before the sample reaches the sequencer.
The first step is collection, followed by preservation and sample preparation for analysis. Most scientists overlook those steps, but not being careful might just be skewing the experiment’s results.
...06-02-2026, 10:05 AM
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