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Has anyone tried running beyond the 2x250bp "official" capacity of the MiSeq? We worked with a sequencing collaborator who hacked together a 2x250 run before the reagents were available by unofficially (although aided by Illumina tech support) pooling a 2x150 and a 1x100 cartridge. Any idea if pooling of a 2x250 and a 2x150 is possible, allowing early access to the supposedly upcoming 2x400 run?
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I'm not sure you would want to try that yet.
see posts below.
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2 x 250 runs are not stably working (in our hands) as yet. Perhaps others have had better luck. No problems getting sequence, it is the decline in quality over the later cycles.
Hopefully this is just teething troubles that will get sorted out over time.
If you have the time (and money) to try the 2 x 400, go right ahead. Let us know what happens.Comment
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Is this with a hardware-upgraded instrument? Does going to lower loading densities appear to help?Originally posted by GenoMax View Post
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PhillipComment
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"Illumina are testing 400bp single-end reads in R&D on the MiSeq, with the longest achieved overlapping paired-read of 678bp"
I think I read somewhere that it was around 80% >Q20 @ 400bpComment
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Yes. Our instrument has received the hardware upgrade.Originally posted by pmiguel View Post
Not sure. v.1. runs (2 x 25 bp) are looking fine, it is the v.2. runs (2 x 250 bp) that seem to be the problem. We have done two long runs so far (one sample and one phiX).Does going to lower loading densities appear to help?
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Phillip
We are working with illumina at the moment. This may be a specific issue with our instrument. We shall see.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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