Tweet
We did some PCR sequencing on Miseq and are trying to see if we could use a stringed together sequence of all amplicons to creat a pseudogenome and input for Miseq reporter in the resequencing option. As we have some internal barcodes in the products, each of our amplicons would have replicates with the different tags at each end. Anyone else has done something similar?
-
-
It should work in theory, but I don't know if the inline barcodes will mess anything up in MSR.You can just try it and report back the results. All you have to do is upload a FASTA file in MCS, then use assign your samples to use it as the genome in MSR. You can requeue the analysis as many times as you want until you get things working just right. -
It does work! Now the next step would be to see if an annotation file can be created for the pseudo genome so that the Miseq reporter can put out the results with annotation. The annotation would be the identity. Could anyone shed some light on how to annotate a psedogenome...Comment
-
Is the fasta header not informative enough? You could also make a bed file that contains the identity information.Comment
-
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
Comment