Combining/multiplexing Nextera and XT indexes

auzzie599 · 10-09-2017, 09:59 AM

Hello,
I am currently using Nextera DNA kits (96 samples, FC-121-1031) to prepare my genomic DNA libraries. I want to be able to multiplex more than 96 individual samples, however.

To do this, I plan to purchase the Nextera XT Index kit v2 'set D' (FC-131-2004), which includes adapter oligos (96 possible combinations for dual indexing) that are unique to the adapters that come with my standard Nextera DNA kit. Combining the two index sets would give me 40 total unique oligos (16 i5 adapters, and 24 i7 adapters, for total of 384 unique combinations) between the two kits.

Does anyone have experience combining standard Nextera DNA indexes with Nextera XT indexes? According to Illumina, they can't validate using XT indexes with the Nextera kit, though they say it generally works fine. What I want to know is, can I successfully combine the indexes from the different kits (i.e. have individual samples with a Nextera i5 index on one end, and an XT i7 index on the other), in order to multiplex 384 samples? I worry that because the XT kits deal with generally lower concentrations/volumes, that the adapter concentrations between the two kits may not match, although there are people on here who say the indexes from the two kits are interchangeable.

Any advice is appreciated!

nucacidhunter · 10-09-2017, 01:06 PM

It works regardless of concentration differences because Nextera kit PCR is done with 4 primers. Two are index primers and other two bind to flow cell binding motif of index primers.

auzzie599 · 10-10-2017, 06:14 AM

Quote:

Originally Posted by nucacidhunter

It works regardless of concentration differences because Nextera kit PCR is done with 4 primers. Two are index primers and other two bind to flow cell binding motif of index primers.

I'm not sure I understand. Can you elaborate on why the 4-primer PCR protocol in the nextera kit would make the concentration of the index primers irrelevant? I am planning to multiplex nextera indexes together with nextera XT indexes... I suppose I won't know if it works until I prep some libraries...

nucacidhunter · 10-10-2017, 07:39 AM

In Nextera protocol tagmented DNA PCR reaction contains index primers/adapters and PPC. Index primers bind to overhangs from tagmentation oligo and fragments with full length adapters is synthesised. Index primers quantity is not enough for whole amplification cycles so PPC oligos bind to 5' end of adapter or library fragments for further amplification. PPC sequences are short and complementary to 5' end of index adapters so it reduces the cost.

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10-09-2017, 09:59 AM	#1
auzzie599 Junior Member Location: Pennsylvania Join Date: Sep 2017 Posts: 6	Combining/multiplexing Nextera and XT indexes Hello, I am currently using Nextera DNA kits (96 samples, FC-121-1031) to prepare my genomic DNA libraries. I want to be able to multiplex more than 96 individual samples, however. To do this, I plan to purchase the Nextera XT Index kit v2 'set D' (FC-131-2004), which includes adapter oligos (96 possible combinations for dual indexing) that are unique to the adapters that come with my standard Nextera DNA kit. Combining the two index sets would give me 40 total unique oligos (16 i5 adapters, and 24 i7 adapters, for total of 384 unique combinations) between the two kits. Does anyone have experience combining standard Nextera DNA indexes with Nextera XT indexes? According to Illumina, they can't validate using XT indexes with the Nextera kit, though they say it generally works fine. What I want to know is, can I successfully combine the indexes from the different kits (i.e. have individual samples with a Nextera i5 index on one end, and an XT i7 index on the other), in order to multiplex 384 samples? I worry that because the XT kits deal with generally lower concentrations/volumes, that the adapter concentrations between the two kits may not match, although there are people on here who say the indexes from the two kits are interchangeable. Any advice is appreciated!

10-09-2017, 01:06 PM	#2
nucacidhunter Senior Member Location: Melbourne Join Date: Jan 2013 Posts: 1,166	It works regardless of concentration differences because Nextera kit PCR is done with 4 primers. Two are index primers and other two bind to flow cell binding motif of index primers.

10-10-2017, 07:39 AM	#4
nucacidhunter Senior Member Location: Melbourne Join Date: Jan 2013 Posts: 1,166	In Nextera protocol tagmented DNA PCR reaction contains index primers/adapters and PPC. Index primers bind to overhangs from tagmentation oligo and fragments with full length adapters is synthesised. Index primers quantity is not enough for whole amplification cycles so PPC oligos bind to 5' end of adapter or library fragments for further amplification. PPC sequences are short and complementary to 5' end of index adapters so it reduces the cost.