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Moving this up to a new thread for more visibility... . We cloned and Sanger sequenced some standard genomic PE libraries and are seeing truncation of the adapters from the 5' end in some libraries, as well as a larger number of molecules containing full PE1 sequences than full PE2 sequences. We used NEB library preparation kits and some custom barcoded adapters from Invitrogen, which contained the phosphothiorate modification and were purified using desalting. Following triplicate 4 cycle PCR (we also tested 8 and 12 cycle PCR but saw little difference in this respect), we cloned several of the libraries into the Fermentas blunt end pJet vector and Sanger sequenced several transformants. While some clones looked normal, several had truncations off the 5' end of PE1 (see attachments). These truncated sequences did contain some bases from the PCR primer on both sides, meaning that we weren't just sequencing unamplified ligated molecules, and there were no adapter-only clones. I haven't been able to come up with a reason why the primers themselves should be truncated aside from being poorly synthesized- but that surprises me since I have not had similar problems with Invitrogen oligos in the past. We are not sure what steps to take next- should we just qPCR quantify the libraries with Kapa so we know how many correct molecules are present and use that to estimate loading amounts? Or did we go wrong elsewhere in library prep?
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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