Let me say upfront that we do Ti amplicon sequencing (pyrotags) on environmental samples using LibA and amplicon processing pipeline/rCafie correction/correct filter settings. Our protocols require reads over 400 nt and min 800K reads passing filter per run. Please don't suggest that we switch to shotgun processing, etc.
What I hope you'll do is join me in demanding that Roche stop selling us poor quality reagents!
Since early December, roughly coinciding with new apyrase formulation, we have had unacceptably low run yields. While a few of these were attributable to underloading, most were not. We include a control amplicon on each region and the yields from it tracked with the rest, countering GS support's diagnosis of excessive short fragments in our amplicon pools (many of you have heard that explanation).
We were also told that the runs passed spec when put through the shotgun processing. As I said, this isn't an acceptable option.
We've been in this game since the start. It was maddening to have tech support imply that we'd become incompetent. I escalated the complaint and our FAS took it seriously, although the levels above her didn't seem to share the concern.
Turns out that Roche's standards for sequencing reagent quality have been lowered. Less than half of the kits that are sold to us are of adequate quality to keep the read signal out of the noise as the read gets up to 400 nt. So yeah, we can get over a million short reads of average length 230 nt. Our FAS looked at kit QC metrics for our good and bad runs--picture attached. Kits can range from 200-600 and pass QC; we need those that are 430-600.
Here's her response:
I don't see how Roche can claim to still support amplicon sequencing or why they bother to maintain an amplicon processing pipeline. I infer from the stock answers given by GS support and the last line of the quoted text that they are NOT going to do either for much longer.
What I hope you'll do is join me in demanding that Roche stop selling us poor quality reagents!
Since early December, roughly coinciding with new apyrase formulation, we have had unacceptably low run yields. While a few of these were attributable to underloading, most were not. We include a control amplicon on each region and the yields from it tracked with the rest, countering GS support's diagnosis of excessive short fragments in our amplicon pools (many of you have heard that explanation).
We were also told that the runs passed spec when put through the shotgun processing. As I said, this isn't an acceptable option.
We've been in this game since the start. It was maddening to have tech support imply that we'd become incompetent. I escalated the complaint and our FAS took it seriously, although the levels above her didn't seem to share the concern.
Turns out that Roche's standards for sequencing reagent quality have been lowered. Less than half of the kits that are sold to us are of adequate quality to keep the read signal out of the noise as the read gets up to 400 nt. So yeah, we can get over a million short reads of average length 230 nt. Our FAS looked at kit QC metrics for our good and bad runs--picture attached. Kits can range from 200-600 and pass QC; we need those that are 430-600.
Here's her response:
"Thank you for the run-reports encompassing all your runs, good and bad. In looking at different metrics for ATGC control beads (Type II), there is a threshold to the signal per base and initial PPi that correlates to your criteria for acceptable run with your current filter parameters in amplicon processing.
A run exhibiting metrics of ATGC ~<430 for the initial PPi and ~<400 for signal per base have excessive Too Short Quality filtering with the amplicon pipeline. Though some sequencing kits meet this criteria, the current QC specification set by 454 Life Sciences for an XLR70 kit to "Pass", has a signal per base of 200 to ~600.
The data (much like your spreadsheet) was submitted to 454 on Monday. Graphing out the trend and asking for ideas of how to boost your signal. Less apyrase in washing enzyme beads has increased signal in the past. Or even using the new XL+ enzyme beads to obtain the higher signal.
But 454 may have other ideas too in pointing out that the filtering parameters for your amplicon sequencing using XLR70 have higher QC metrics than the shotgun pipeline."
A run exhibiting metrics of ATGC ~<430 for the initial PPi and ~<400 for signal per base have excessive Too Short Quality filtering with the amplicon pipeline. Though some sequencing kits meet this criteria, the current QC specification set by 454 Life Sciences for an XLR70 kit to "Pass", has a signal per base of 200 to ~600.
The data (much like your spreadsheet) was submitted to 454 on Monday. Graphing out the trend and asking for ideas of how to boost your signal. Less apyrase in washing enzyme beads has increased signal in the past. Or even using the new XL+ enzyme beads to obtain the higher signal.
But 454 may have other ideas too in pointing out that the filtering parameters for your amplicon sequencing using XLR70 have higher QC metrics than the shotgun pipeline."