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I am planning on performing an illumina multiplex sequencing run on a library of sequences that I have compiled. They are digested out of plasmid and consist of ~10 unvaried nucleotides, 60 varied nucleotides, ~40 unvaried nucleotides, 60 varied nucleotides, then 10 unvaried nucleotides in that order. I have seen in previous papers that there are some issues with the cluster identification step for sequences which are unvaried in their first few nucleotides. Is this still a major problem? Is there a way to get around this without adding extra varied nucleotides to the ends of my sequences? Or is this the only robust solution?
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You need to design a custom sequencing primer.
Use the sequence of the adapter+sequence of your non-variable region(10 bases)
Then create a sequencing primer that is complementary to the last base of your nonvariable region-then work back.
When the Tm of the primer is above 65 (that way it will work on both HiSeq and MiSeq) you have that primer synthesized and HPLC purified.
Use the primer at 100uM.
Whoever is doing you sequencing will know how to use it. -
If you are sequencing a small number of plasmid-derived inserts, then you are likely to not need the full number of reads in a lane. An alternative to creating a custom primer is to just spike your library into a control lane of PhiX on a HiSeq, or mix in a higher-complexity library (usually someone has something they want a little more sequence for) on a MiSeq. It seems like the recent software changes have made the MiSeq less sensitive to complexity reductions anyway.Providing nextRAD genotyping and PacBio sequencing services. http://snpsaurus.comComment
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Please can your help in any library for plasmodium gametocytes. Thnaks . ErnyComment
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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.07-01-2026, 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...
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