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Christina - Those enrichment numbers seem a little high to me, so I would keep washing. As for my problem, it seemed to work itself out when I went from 5 copies/bead down to 2 copies/bead during amplicon sequencing (did not do a titration).
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Christina85... I am ABSOLUTELY sure it was the pipettor. I have photos and data to support this is a side-by-side experiment we did. We checked everything before hand as well... pcr plate, thermal cyclers, caps for the pcr plates, etc and we had Roche is 3 times. It wasn't until 454 came in with Roche and made this recommendation - now everything works like a dream. And there is correlations between our SVs and LVs (almost perfect). I can send you data / photos if you want to see them. Email me at [email protected].Comment
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One thing I've been doing to increase accuracy (I hope) is perform 2 volume measurements and 2 bead counts for each tube, and then use the averages. Measuring ~1200ul with a pipette is pretty tricky, and I've found that 2 back-to-back measurements, with the same pipette, tip, and user can give differences up to 50ul, and two back-to-back bead counts (using different Accuvettes and different 3ul aliquots) can give differences up to 500,000 beads. Little deviations like these can certainly add up and make troubleshooting difficult, so I'm doing it all in duplicate.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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