RTA - bug - other options

Elsie · 06-03-2014, 02:57 PM

Hi, it appears that there is a bug in the RTA software (we are running RTA1.18.54). We are running some 16S on the MiSeq and the % > Q30 charts, we often see a sharp drop off right at the beginning of the read. Illumina techsupport have indicated that this is due to the RTA software, where it could not adjust the signal from the ATGC. RTA is involved in the primary analysis and appears crucial for generating the BCL files, does anyone know if it is possible to sidestep RTA or any other ways to compensate for this issue?
thanks.

Elsie · 06-04-2014, 02:34 PM

A further reply from Illumina:

"Why do we see a drop in Q scores in the initial cycles of read 1? This is characteristic of runs which use our suggested V3/V4 target primers, and appears to be linked to a specific motif, though we don’t quite understand the mechanism by which signal to noise is so reduced in these initial cycles. We considered revising the design before releasing the demonstrated protocol but what we found is that this didn’t significantly impact the pass filter rates or data quality, and since it happens in the primer sequence its not affecting the part of the read used in the analysis. This means that although it looks alarming, the functional impact is essentially nil. Customers running this assay will see a very different run profile in SAV as they are accustomed to just because it is a single amplicon low diversity run."

So maybe no need to bypass RTA but something to be aware of.

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06-03-2014, 02:57 PM	#1
Elsie Member Location: Australia Join Date: Mar 2011 Posts: 85	RTA - bug - other options Hi, it appears that there is a bug in the RTA software (we are running RTA1.18.54). We are running some 16S on the MiSeq and the % > Q30 charts, we often see a sharp drop off right at the beginning of the read. Illumina techsupport have indicated that this is due to the RTA software, where it could not adjust the signal from the ATGC. RTA is involved in the primary analysis and appears crucial for generating the BCL files, does anyone know if it is possible to sidestep RTA or any other ways to compensate for this issue? thanks. Last edited by Elsie; 06-03-2014 at 03:06 PM. Reason: wrong version listed

06-04-2014, 02:34 PM	#2
Elsie Member Location: Australia Join Date: Mar 2011 Posts: 85	A further reply from Illumina: "Why do we see a drop in Q scores in the initial cycles of read 1? This is characteristic of runs which use our suggested V3/V4 target primers, and appears to be linked to a specific motif, though we don’t quite understand the mechanism by which signal to noise is so reduced in these initial cycles. We considered revising the design before releasing the demonstrated protocol but what we found is that this didn’t significantly impact the pass filter rates or data quality, and since it happens in the primer sequence its not affecting the part of the read used in the analysis. This means that although it looks alarming, the functional impact is essentially nil. Customers running this assay will see a very different run profile in SAV as they are accustomed to just because it is a single amplicon low diversity run." So maybe no need to bypass RTA but something to be aware of.