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Someone recently asked me to help them build an assembly for some 454 data sequenced after using the Sigma/Aldrich Genomeplex Whole Genome Amplicification kit. Unfortunately, the primer sequencing used in library construction are all proprietary sequences and I can't seem to find them anywhere. Has anyone else dealt with this type of data and need for unknown primer removal. Any suggestions on how to identify possible primer sequences and cut them out of my sequences aside from some sort of wholesale global end-trimming approach?
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We had a similar issue with some metagenomics samples that we were doing for a collaborator, and if I recall correctly, we could identify the Sigma primers by examining a small number of the reads. The primer sequences were common on all of the reads. -
Thanks for the response, bbeitzel. Yes, I noticed a common sequence as well, which is probably the Universal primer, in lots (~53% of all, ~5% at 3' end and ~48% at 5' end) of the reads, but not all. Unfortunately, the sequence is ~18bp long and Sigma said their universal primer was 30 bp. Was your motif closer to one of these two? Unfortunately, our template in this case is likely to have lots of repetitive motifs in them (microdissected chromosome with big peri-centromeric regions) so we need to be as careful as possible about removing components that are actually meaningful to us.Comment
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Well, for future reference, if anyone runs into this thread, I ended up using an on-line program called TagCleaner to identify possible primer tags in next-gen sequences and then cut them out using that tool. Seemed to work well.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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