Methodological improvements in sequencing methodology that have taken place since the original publication of the data could explain the discrepancy.
Since Dr. Wu first developed the DNA sequencing methodology, and established the primer-extension principle, "father of DNA sequencing" should be a correct title for him.
Here, we describe basic principles, study design, and a workflow of 16S ribosomal RNA gene sequencing methodology, primarily for investigators who are not familiar with this approach.
iAs a result of technological advances (especially the introduction of next-generation sequencing methodology), the single-organism approach has recently succeeded in shifting its emphasis from research on traditional model species to ecologically-interesting species, e.g. the water flea Daphnia Pulex.
We believe that the observed mutant reads reflect minority variants undetectable by bulk sequencing methodology.
Sequencing methodology was used as a second confirmatory method on a subset of samples to verify that correct alleles were being identified by ARMS-PCR.
However, these processes impose additional confounding factors into the data that SMS avoids entirely due to the direct nature of the sequencing methodology.
We show that the integration of DNA-based quantification and large-scale sequencing methodology presented here produces unbiased data and allows the full determination of the whole pool of ligand sequences available after "n" rounds of selection.
Although certain mutations (e.g. rearrangements, large tandem repeat-polymorphisms) are not reliably detected by the sequencing methodology used, it is not unlikely that the few deletions detected completely may explain the attenuation of virulence in the strain FSC043.
The 454 sequencing methodology eliminates the need to construct a hierarchical set of clones for each read fragment as in electrophoresis-based sequencing: it instead amplifies the read fragments on the beads using PCR emulsion.
A third possibility, is that the Pfcrt allele is present at a low fraction in the time 0 sample, too low to be detected (masked by the wild type allele and not detected by the PCR followed by DNA sequencing methodology) and too low to translate into a shifted IC50 in vitro (as these parasites represent a low fraction, they incorporate a low amount of 3H hypoxanthine and remain unnoticed).
