Nextera Flex 1/2 and 1/4 Reaction Experiences?

UCan'tBcereus · 09-21-2018, 09:43 AM

Hi Everyone,

We are about to run through a test of the Nextera Flex kit, and I wanted to see if anyone has had a positive experience cutting down the RXN volume down to 1/2 RXN or 1/4 RXN.

When we use Nextera XT, we cut down to 1/2 RXN and 1/4 RXN successfully with no issues, other than having to experiment a little with DNA input.

I have full confidence that Nextera Flex libraries will work at 1/2 RXN and 1/4 RXN, but I was wondering if anyone has seen how the "no normalization required with >100 ng DNA input" aspect holds up. I.e at 1/2 RXN, has anyone seen that at 50 ng DNA input the library yields are close enough to not require extensive normalization?

Any other Nextera Flex tips that anyone has noticed would be great too. Thanks everyone!

Quick Side Note: After talking to Illumina one of their reps mentioned that 96 Indexes (UDI not combinatorial) will be released sometime next month for Nextera Flex, with 384 UDI indexes being available "early 2019", in case anyone was interested.

ikripp · 09-23-2018, 04:34 PM

We do 1/5 reactions for Flex as we also did for XT before changing over.
They appear to be comparable and we have had no issues.

thang198 · 10-02-2018, 11:11 AM

Quote:

Originally Posted by UCan'tBcereus

Hi Everyone,

We are about to run through a test of the Nextera Flex kit, and I wanted to see if anyone has had a positive experience cutting down the RXN volume down to 1/2 RXN or 1/4 RXN.

When we use Nextera XT, we cut down to 1/2 RXN and 1/4 RXN successfully with no issues, other than having to experiment a little with DNA input.

I have full confidence that Nextera Flex libraries will work at 1/2 RXN and 1/4 RXN, but I was wondering if anyone has seen how the "no normalization required with >100 ng DNA input" aspect holds up. I.e at 1/2 RXN, has anyone seen that at 50 ng DNA input the library yields are close enough to not require extensive normalization?

Any other Nextera Flex tips that anyone has noticed would be great too. Thanks everyone!

Quick Side Note: After talking to Illumina one of their reps mentioned that 96 Indexes (UDI not combinatorial) will be released sometime next month for Nextera Flex, with 384 UDI indexes being available "early 2019", in case anyone was interested.

Hi UCan'tBcereus,
Would you please tell me how much input you used for the 1/2 and 1/4 RXN?
Thank you,

mdegraaf · 02-12-2020, 10:50 AM

Hi I was wondering if you have answered your own question by now. We are doing the Nextera Flex at 1/5 and are wondering about the input and number of cycles.

UCan'tBcereus · 02-14-2020, 11:13 AM

Hello,

We haven't extensively tested the higher input of DNA at this time. The Lab that we do this for will the majority of the time give us very low concentration DNA samples, so often we are doing 1 ng DNA Input and then doing 12 cycles of PCR. Because the DNA samples we get aren't great, we end up having to quant/Norm by hand. We have a liquid handler so quantifying and normalizing libraries is pretty painless for us. We haven't had a large project where we had samples with lots of DNA to play with.

16 cycles seems really aggressive. We have done .375 ng DNA with 12 cycles and gotten low contraption, but usable libraries before.

Personally, if there is plenty of DNA to work with, I would use ~75-80 ng. At Full RXN the range is 100-500 ng (more than that will will cause issues). So at 1/5 RXN then 20-100 ng should be fine. I would shoot under 100 ng that in case your quants are off and you actually use more DNA than you think. I remember talking to our local Illumina rep though and she did say that up to 1000 ng had been done successfully in other labs (of course not supported if it failed...). Since you are doing 1/5 RXNS you won't be supported for failures anyways. For # of cycles, I'm not really sure. I don't have a good idea of how much Library needs to be made to reach saturation.

You should Look into BioRxiv for a "HackFLex" Paper too. There they tried to home-brew Nextera Flex as much as possible, but diluted the BLTs 1:50, used 10 uL DNA, and with 12 cycles the libraries still worked.

I'm still really interested if this will work, so let us know how it ends up working out. I think you will have do a small test and see, even for a few samples (75-80 ng at 6 cycles, then 75-80 ng at 12 cycles). I think anything more than 12 cycles is going to bias the sequencing results more than it needs to be. Good Luck!

mdegraaf · 02-25-2020, 02:46 PM

Hi,
I apologize for the late reply, I was very busy finishing the library prep's in time.
We ended up doing 8 cycles for the higher concentration samples and 12 for lower. The 8 cycles should have provided normalization in the higher concentration samples. I'm not sure what range of output is considered normalized but we ended up doing normalization by hand, which actually wasn't too bad for 90 samples.

We did most of the protocol at 1/5th strength, some wash steps in 1/2 or full. We did do a small test before deciding this and the Bioanalyzer profiles were identical as well as the results from the MiSeq run we did. We did not have time to experiment with number of cycles. Also we are more focused on identification so if results are a bit unbalanced that is ok for our purposes.

We just shipped our samples to be sequenced so it will take some time before we have the results.

mrsrankind1990 · 03-27-2020, 10:26 AM

Quote:

Originally Posted by mdegraaf

Hi,
I apologize for the late reply, I was very busy finishing the library prep's in time.
We ended up doing 8 cycles for the higher concentration samples and 12 for lower. The 8 cycles should have provided biggest variety of essay topics https://www.wowessays.com/topics/ normalization in the higher concentration samples. I'm not sure what range of output is considered normalized but we ended up doing normalization by hand, which actually wasn't too bad for 90 samples.

We did most of the protocol at 1/5th strength, some wash steps in 1/2 or full. We did do a small test before deciding this and the Bioanalyzer profiles were identical as well as the results from the MiSeq run we did. We did not have time to experiment with number of cycles. Also we are more focused on identification so if results are a bit unbalanced that is ok for our purposes.

We just shipped our samples to be sequenced so it will take some time before we have the results.

Hello. Any updates about results?

Godzilla · 05-01-2020, 10:23 AM

Quote:

Originally Posted by ikripp

We do 1/5 reactions for Flex as we also did for XT before changing over.
They appear to be comparable and we have had no issues.

Can you post the protocol you are using to achieve 1/5X on the kit?

E

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09-21-2018, 09:43 AM	#1
UCan'tBcereus Member Location: New England Join Date: Aug 2018 Posts: 12	Nextera Flex 1/2 and 1/4 Reaction Experiences? Hi Everyone, We are about to run through a test of the Nextera Flex kit, and I wanted to see if anyone has had a positive experience cutting down the RXN volume down to 1/2 RXN or 1/4 RXN. When we use Nextera XT, we cut down to 1/2 RXN and 1/4 RXN successfully with no issues, other than having to experiment a little with DNA input. I have full confidence that Nextera Flex libraries will work at 1/2 RXN and 1/4 RXN, but I was wondering if anyone has seen how the "no normalization required with >100 ng DNA input" aspect holds up. I.e at 1/2 RXN, has anyone seen that at 50 ng DNA input the library yields are close enough to not require extensive normalization? Any other Nextera Flex tips that anyone has noticed would be great too. Thanks everyone! Quick Side Note: After talking to Illumina one of their reps mentioned that 96 Indexes (UDI not combinatorial) will be released sometime next month for Nextera Flex, with 384 UDI indexes being available "early 2019", in case anyone was interested.

09-23-2018, 04:34 PM	#2
ikripp Member Location: QLD, Aus Join Date: Jan 2018 Posts: 19	We do 1/5 reactions for Flex as we also did for XT before changing over. They appear to be comparable and we have had no issues.

02-12-2020, 10:50 AM	#4
mdegraaf Junior Member Location: Canada Join Date: Feb 2020 Posts: 5	Hi I was wondering if you have answered your own question by now. We are doing the Nextera Flex at 1/5 and are wondering about the input and number of cycles.

02-14-2020, 11:13 AM	#5
UCan'tBcereus Member Location: New England Join Date: Aug 2018 Posts: 12	Hello, We haven't extensively tested the higher input of DNA at this time. The Lab that we do this for will the majority of the time give us very low concentration DNA samples, so often we are doing 1 ng DNA Input and then doing 12 cycles of PCR. Because the DNA samples we get aren't great, we end up having to quant/Norm by hand. We have a liquid handler so quantifying and normalizing libraries is pretty painless for us. We haven't had a large project where we had samples with lots of DNA to play with. 16 cycles seems really aggressive. We have done .375 ng DNA with 12 cycles and gotten low contraption, but usable libraries before. Personally, if there is plenty of DNA to work with, I would use ~75-80 ng. At Full RXN the range is 100-500 ng (more than that will will cause issues). So at 1/5 RXN then 20-100 ng should be fine. I would shoot under 100 ng that in case your quants are off and you actually use more DNA than you think. I remember talking to our local Illumina rep though and she did say that up to 1000 ng had been done successfully in other labs (of course not supported if it failed...). Since you are doing 1/5 RXNS you won't be supported for failures anyways. For # of cycles, I'm not really sure. I don't have a good idea of how much Library needs to be made to reach saturation. You should Look into BioRxiv for a "HackFLex" Paper too. There they tried to home-brew Nextera Flex as much as possible, but diluted the BLTs 1:50, used 10 uL DNA, and with 12 cycles the libraries still worked. I'm still really interested if this will work, so let us know how it ends up working out. I think you will have do a small test and see, even for a few samples (75-80 ng at 6 cycles, then 75-80 ng at 12 cycles). I think anything more than 12 cycles is going to bias the sequencing results more than it needs to be. Good Luck!

02-25-2020, 02:46 PM	#6
mdegraaf Junior Member Location: Canada Join Date: Feb 2020 Posts: 5	Hi, I apologize for the late reply, I was very busy finishing the library prep's in time. We ended up doing 8 cycles for the higher concentration samples and 12 for lower. The 8 cycles should have provided normalization in the higher concentration samples. I'm not sure what range of output is considered normalized but we ended up doing normalization by hand, which actually wasn't too bad for 90 samples. We did most of the protocol at 1/5th strength, some wash steps in 1/2 or full. We did do a small test before deciding this and the Bioanalyzer profiles were identical as well as the results from the MiSeq run we did. We did not have time to experiment with number of cycles. Also we are more focused on identification so if results are a bit unbalanced that is ok for our purposes. We just shipped our samples to be sequenced so it will take some time before we have the results.