RTA v2.8 : Conflicts with low complexity sequence

nickp · 10-13-2010, 11:54 AM

We routinely use an in-line barcoding system with bases 1-4 as variable barcode nucleotides and base 5 is a mandatory T in all indexed libraries.
We have run several such libraries under RTA 2.6 using a PhiX lane to call thresholds. An Illumina tech support suggested this might now be a problem with RTA v2.8 as it used the first 5 nucleotides to threshold rather than the first 4 that RTA2.6 used. Even with a PhiX lane for thresholding they are worried that our low complexity start to all our reads might now cause a conflict.

Anyone else using low complexity reads in RTA 2.8 ?. Any problems over RTA 2.6 ?

Elsie · 06-03-2014, 04:03 PM

Hi Nickp,
A little bit late but 2.8 sounds wrong, the current versions are only 1.9
http://support.illumina.com/sequenci...downloads.ilmn
cheers.

thomasblomquist · 06-04-2014, 10:19 AM

This is indeed concerning. We synthesized our in-line barcoding reagents to have the first 4 bases be randomized followed by our less complex sequences.

Let us all know if you have any issues going forward with the 4 base randomization and the new RTA?

-Tom Blomquist

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10-13-2010, 11:54 AM	#1
nickp Junior Member Location: Oxford Join Date: Oct 2010 Posts: 1	RTA v2.8 : Conflicts with low complexity sequence We routinely use an in-line barcoding system with bases 1-4 as variable barcode nucleotides and base 5 is a mandatory T in all indexed libraries. We have run several such libraries under RTA 2.6 using a PhiX lane to call thresholds. An Illumina tech support suggested this might now be a problem with RTA v2.8 as it used the first 5 nucleotides to threshold rather than the first 4 that RTA2.6 used. Even with a PhiX lane for thresholding they are worried that our low complexity start to all our reads might now cause a conflict. Anyone else using low complexity reads in RTA 2.8 ?. Any problems over RTA 2.6 ?

06-03-2014, 04:03 PM	#2
Elsie Member Location: Australia Join Date: Mar 2011 Posts: 85	Hi Nickp, A little bit late but 2.8 sounds wrong, the current versions are only 1.9 http://support.illumina.com/sequenci...downloads.ilmn cheers.

06-04-2014, 10:19 AM	#3
thomasblomquist Member Location: Ohio Join Date: Jul 2012 Posts: 68	This is indeed concerning. We synthesized our in-line barcoding reagents to have the first 4 bases be randomized followed by our less complex sequences. Let us all know if you have any issues going forward with the 4 base randomization and the new RTA? -Tom Blomquist