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low Q30 in index, miseq v3 600
I recently ran a v3 600 cycle kit on a Miseq and the Q30 values dropped way down in the index read. The sample was 2-plex using N701 and N702. The basecalls in the index fastq look like junk. Any ideas as to what the problem could be? I'm using the standard primers.
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You are using one of the recommended tag combinations for 2-plex libraries so that part should be ok. So you were not able to demultiplex the data (are there lots of N's in tag reads? Have you talked with tech support? -
One possible issue would be design or oligo manufacturing fault if you have not used actual Illumina Nextera kit for library prep which seems to be the case. I wonder what was the %reads identified.Comment
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I had the same issue, but in my case custom sequencing primers were used. Tech support suggested the issue is coming from the primers, but previous runs were just fine. My % reads identified was 11 %. Pretty low. Any thoughts?Comment
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.Today, 11:43 AM
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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... -
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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