Hi,
We previously performed a 454 amplicon sequencing run on our gene library with great results back in March '12. We prepared a new amplicon library of the same gene essentially the same way as before only this time with more MID barcodes. The core facility we are working with has repeated the run twice now with very poor results and they can't figure out what seems to be going wrong nor has Roche tech support been able to figure out what the problem is.
Some details: amplicons created with lib-A A/B fusion primers, gel purified, and AMPure XP bead purified. Running on the fragment analyzer shows a clean band at 421 bp (as expected, though a little high, amplicons should be 398-412 bp). There is very small double peak at ~30 bp, but this is too small to be primers or primer dimers, and may be an artifact of the lower marker. The %bead enrichment was around 12-15%.
After the first run, the Raw Wells was 2,472,168 and the Key Pass wells was 2,388,051 both of which look good. The dot filter then removed 169,004 and the mixed filter removed 256,456 (total of 18%). However the Short Quality filter then removed 1,835,645 (77%) of reads, such that only 125,726 passed filter (5%). A second run had essentially the same results but even lower pass filter (4%).
Roche was looking at the sequences and said they didn't see major homopolymer issues or flashing interference problems and that the control beads looked good.
Of the good reads we have received, the sequences look good and we see a ditribution of the MID barcodes as we would expect. So we are frustrated and at a loss to figure out what is going wrong with these runs. One difference between these runs and the previous successful one is our core manager informed us that Roche changed their beads to amplify better.
Has anyone had such issues with low pass filter/short reads? Any ideas or suggestions as to what may be going wrong here would be appreciated.
Thank you!
We previously performed a 454 amplicon sequencing run on our gene library with great results back in March '12. We prepared a new amplicon library of the same gene essentially the same way as before only this time with more MID barcodes. The core facility we are working with has repeated the run twice now with very poor results and they can't figure out what seems to be going wrong nor has Roche tech support been able to figure out what the problem is.
Some details: amplicons created with lib-A A/B fusion primers, gel purified, and AMPure XP bead purified. Running on the fragment analyzer shows a clean band at 421 bp (as expected, though a little high, amplicons should be 398-412 bp). There is very small double peak at ~30 bp, but this is too small to be primers or primer dimers, and may be an artifact of the lower marker. The %bead enrichment was around 12-15%.
After the first run, the Raw Wells was 2,472,168 and the Key Pass wells was 2,388,051 both of which look good. The dot filter then removed 169,004 and the mixed filter removed 256,456 (total of 18%). However the Short Quality filter then removed 1,835,645 (77%) of reads, such that only 125,726 passed filter (5%). A second run had essentially the same results but even lower pass filter (4%).
Roche was looking at the sequences and said they didn't see major homopolymer issues or flashing interference problems and that the control beads looked good.
Of the good reads we have received, the sequences look good and we see a ditribution of the MID barcodes as we would expect. So we are frustrated and at a loss to figure out what is going wrong with these runs. One difference between these runs and the previous successful one is our core manager informed us that Roche changed their beads to amplify better.
Has anyone had such issues with low pass filter/short reads? Any ideas or suggestions as to what may be going wrong here would be appreciated.
Thank you!
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