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I am preparing the library for single end RNA-seq following the illumina protocol. However, by agilent analysis for library, the two smear bands occur at 200bp and 500 bp. I do not know why the 500 bp band is visible. Who has the same experience? Anyone's idea for this result?
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What stage of the library prep are you at?? Is this the final purified library? What size bands are you selection? -
It is purified library for subsequent cluster generation in mRNA-seq. This library is generated by PCR enrichment during which the gel-seclection size at 200 bp is used as template. The PCR cycle is 15. Thus, in theory and according to illumina protocol of sample preparation for single end RNA-seq, the band shoud be only at around 200 bp, and the 500 bp band should not occur. I do not know why 500 bp band is yielded? I need 200 bp band.Originally posted by peromhc View PostComment
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What about running another gel and selecting only the size you want?Comment
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Poly, I would follow LMc's advice and do another round of size selection- it's hard to know what that band might be, but Im pretty sure you dont want it!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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