TruSeq v3 Cluster Kits with TruSeq SBS kits?

rgejman · 07-12-2011, 04:08 PM

Hi all,

Has anyone experimented with using TruSeq SBS kits (i.e. not v3 SBS kits) to sequence TruSeq v3 Cluster Kits and flow cells?. I have heard through the grapevine that it can be done and I was hoping that this might resolve a cluster kit / SBS kit mismatch problem.

Thanks!

GW_OK · 07-13-2011, 06:51 AM

Didja try calling Illumina tech support?

bkingham · 07-13-2011, 07:21 AM

I haven't done it... but did ask the question. It can be done with no problem.
The v3 cluster kit advancement is more reads (bigger flow cell/more clustering real estate), and improved GC resolution (chemistry changes). The v3 sequencing kit advancement is simply better stability, you can load 209 cycles of ICR, rather than split for a 2x101. TruSeq v1 seq kits can be used with v3 cluster kits with no reduction in q scores (presumably), you just have to split ICR for a 2x101

pmiguel · 07-13-2011, 08:28 AM

Are there not issues with switching from v1 -> v3 requiring an initial "double priming" of fluids lines that blows an extra 4 cycles worth of reagents? I guess if you have not run v3 chemistry yet it doesn't matter. But if you were thinking about switching back and forth then this could be an issue.

--
Phillip

rgejman · 07-13-2011, 11:07 AM

Does this "double priming" have to happen once or every time you load a v1 vs. v3 flow cell? I actually buy 200 cycle kit (actually 202 cycle kits) and aliquot them into 4x 50 cycle kits (for which I can actually run 51 cycles—works like a charm). If double priming happens every time I switch then this will not be feasible...

pmiguel · 07-13-2011, 11:33 AM

It is the SBS kit, not the flowcell that is the issue.

From what we have been told, a double prime needs to be done every time one uses v3 chemistry in an instrument that used v1 chemistry for its previous run.

The 200 cycle kits are supposed to be sufficient for a 4 (single prime)+ 101+ 7 +101 -- so roughly 213 cycles worth. A 50 cycle kit would (by the same logic) be 4 + 50 +7 = 61 cycles worth.

--
Phillip

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07-12-2011, 04:08 PM	#1
rgejman Junior Member Location: New York Join Date: Nov 2010 Posts: 4	TruSeq v3 Cluster Kits with TruSeq SBS kits? Hi all, Has anyone experimented with using TruSeq SBS kits (i.e. not v3 SBS kits) to sequence TruSeq v3 Cluster Kits and flow cells?. I have heard through the grapevine that it can be done and I was hoping that this might resolve a cluster kit / SBS kit mismatch problem. Thanks!

07-13-2011, 06:51 AM	#2
GW_OK Senior Member Location: Oklahoma Join Date: Sep 2009 Posts: 411	Didja try calling Illumina tech support?

07-13-2011, 07:21 AM	#3
bkingham Member Location: delaware Join Date: May 2008 Posts: 11	I haven't done it... but did ask the question. It can be done with no problem. The v3 cluster kit advancement is more reads (bigger flow cell/more clustering real estate), and improved GC resolution (chemistry changes). The v3 sequencing kit advancement is simply better stability, you can load 209 cycles of ICR, rather than split for a 2x101. TruSeq v1 seq kits can be used with v3 cluster kits with no reduction in q scores (presumably), you just have to split ICR for a 2x101

07-13-2011, 08:28 AM	#4
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	Are there not issues with switching from v1 -> v3 requiring an initial "double priming" of fluids lines that blows an extra 4 cycles worth of reagents? I guess if you have not run v3 chemistry yet it doesn't matter. But if you were thinking about switching back and forth then this could be an issue. -- Phillip

07-13-2011, 11:07 AM	#5
rgejman Junior Member Location: New York Join Date: Nov 2010 Posts: 4	Does this "double priming" have to happen once or every time you load a v1 vs. v3 flow cell? I actually buy 200 cycle kit (actually 202 cycle kits) and aliquot them into 4x 50 cycle kits (for which I can actually run 51 cycles—works like a charm). If double priming happens every time I switch then this will not be feasible...

07-13-2011, 11:33 AM	#6
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	It is the SBS kit, not the flowcell that is the issue. From what we have been told, a double prime needs to be done every time one uses v3 chemistry in an instrument that used v1 chemistry for its previous run. The 200 cycle kits are supposed to be sufficient for a 4 (single prime)+ 101+ 7 +101 -- so roughly 213 cycles worth. A 50 cycle kit would (by the same logic) be 4 + 50 +7 = 61 cycles worth. -- Phillip