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Hello, my ddRAD libraries (mean 550 pb) contain the first 5 identical bases at their ends, belonging to the partial restriction site. They will be paired-end sequenced in a MiSeq. I would like to modify the Illumina sequencing primers by adding those 5 bases at the 3 prime end of each primer. Thus, a higher diversity is expected in the first sequenced bases and also an adequate cluster generation. Does anyone have experience with this approach? and the use or not, and its PhiX concentration. Thank you. Best regards.
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It could work - however you will not be able to sue the PhiX libraries as spike in since you will can't use the Illumina sequencing primers together with your extended versions. -
Thank you. It is right. So the question is what can work best. 1) Use modified primers to bypass the restriction site or 2) use a high concentration of PhiX (how much?).Comment
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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.
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Introduction
Single-cell sequencing technologies have undergone remarkable advances over the past decade, transitioning from low-throughput experimental approaches to highly scalable platforms capable of...05-22-2026, 06:42 AM
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