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Old 07-21-2016, 07:44 AM   #1
cerbatana
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Red face Nextera Mate Pair library: expected size after tagmentation

Hi! I'm having problems with the tagment genomic DNA step of the Nextera mate-pair library preparation kit and I really don't know if the enzyme is working at all. I' attaching the bioanalyzer DNA 12000 results right after the tagment genomic DNA step. I tested two different lots of the enzyme but the results are the same, and I also don't think is a DNA quality problem because this DNA has been successfully used with the Nextera XT kit which also uses a tagmentation step.

I used 4 ug of DNA as input (good quality according to PFGE results with fragments longer than 50 kbp) and 4 uL (lane 1) or 8 uL (lane 2) of tagment enzyme from one lot and 4 uL from another lot (lane 3). Lane 4 was loaded with water.

There seems to be something in the 17000 bp region but I don't know if this long fragmented DNA or unfragmented DNA that migrated with the longer band of the ladder.

Can somebody please explain what happened with my fragmented DNA? Thanks!
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Old 07-21-2016, 01:51 PM   #2
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You need to clean tagmented DNA before BA run for correct sizing because Nextera nzyme still would be attached to DNA causing abarrent migration in the Chip. Tagmented DNA should have a peak at 8-10kb. Larger peaks are indicator of inhibitor presence in sample and a clean up should remove most of those. I am assuming that the kit reagents are fine.
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Old 07-21-2016, 02:54 PM   #3
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In addition to what nucacidhunter wrote, HMW DNA can be difficult to quantify and you might still be overloading the reaction.
Did you use any CTAB during the DNA prep?
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Old 07-21-2016, 04:13 PM   #4
cerbatana
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Hi! I cleaned the tagmented dna with the zymo columns and what I applied to the dna chip was diluted according to the protocol in 7 uL of water. Is it necessary an additional clean up step?

On the other hand my DNA was isolated using the wizard DNA purification kit and quantified using Qubit.
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Old 07-21-2016, 04:49 PM   #5
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Dilution of clean tagmented DNA in 7 ul of water is to dilute for 12K Chip even though it is recommended by the user guide. I would suggest Qubit quantifying clean tagmented DNA and input and running equal quantity side by side to see if tagmentation has been successful.

My suggestions:
1- Try another DNA sample in a 1/10 tagmentation reaction to check reagents.
2- If reagents are OK try cleaning input DNA with Zymo columns and use RSB, EB or TE0.1 for elution (not the kit elution buffer).
3- Set up fresh tagmentaion reaction and increase Tagment enzyme by 25% (15ul).
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Old 07-24-2016, 09:34 PM   #6
cerbatana
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Quote:
Originally Posted by nucacidhunter View Post
Dilution of clean tagmented DNA in 7 ul of water is to dilute for 12K Chip even though it is recommended by the user guide. I would suggest Qubit quantifying clean tagmented DNA and input and running equal quantity side by side to see if tagmentation has been successful.

My suggestions:
1- Try another DNA sample in a 1/10 tagmentation reaction to check reagents.
2- If reagents are OK try cleaning input DNA with Zymo columns and use RSB, EB or TE0.1 for elution (not the kit elution buffer).
3- Set up fresh tagmentaion reaction and increase Tagment enzyme by 25% (15ul).
Thanks a lot for your help. I was also told to try using TapeStation since Bioanalyzer could not render reliable results some times with fragments of these sizes (gel migration problems I assume) so I thought about using pulse field gel electrophoresis to check size distribution.

Regarding your suggestions I always use RSB at room temperature to elute the tagmented DNA. I will try using the different amounts of reactions as you suggested and I'll post the result.
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Old 07-24-2016, 10:42 PM   #7
nucacidhunter
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Quote:
Originally Posted by cerbatana View Post
Thanks a lot for your help. I was also told to try using TapeStation since Bioanalyzer could not render reliable results some times with fragments of these sizes (gel migration problems I assume) so I thought about using pulse field gel electrophoresis to check size distribution.
To me it seems that tagmentation has been less efficient than it should be. I was reluctant to comment on it because I have not seen the input DNA size distribution. 8-10 kb fragments from tagmentation step in my experience is resolve well on 12kb Chip. It can be resolved on gDNA ScreenTape as well but the observed size is dependent on recovery amount and is not as accurate as 12K Chip.
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Old 07-25-2016, 02:05 AM   #8
cerbatana
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Quote:
Originally Posted by nucacidhunter View Post
To me it seems that tagmentation has been less efficient than it should be. I was reluctant to comment on it because I have not seen the input DNA size distribution. 8-10 kb fragments from tagmentation step in my experience is resolve well on 12kb Chip. It can be resolved on gDNA ScreenTape as well but the observed size is dependent on recovery amount and is not as accurate as 12K Chip.
Thank you for the feedback. I was also thinking about the enzyme not being efficient enough, but in two different batches I saw it as a little bit unlikely. The funny thing is that I showed these results to Illumina support and they said that this size distribution (all recovered tagmented DNA in the largest size region of the chip gel) is expected when using 4 uL or 8 uL of tagmentation enzyme, so it is safe to continue with the protocol according to them. I will run PFGE with tagmented DNA to check the size anyway.

By the way, the protocol says that tagmented samples can be stored for no more than 24 hours at -20 degrees, by this point my treated samples completed 4 days of storage. Has anyone tried using samples stored for more than the recommended time after tagmentation? it's double stranded DNA so It should be stable right?
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Old 07-25-2016, 04:14 AM   #9
nucacidhunter
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Tech support is right. I thought that you have used 12 ul of enzyme as recommended. I have used frozen tagmented fragments but cannot remember if it was after tagmentation or strand displacement.

Tagmentation enzyme is one of the limiting reagents and you already have used DNA as well. So, there is no harm continuing the protocol. If library yields is not enough then you may prepare another one.
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Old 07-25-2016, 10:11 PM   #10
cerbatana
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Quote:
Originally Posted by nucacidhunter View Post
Tech support is right. I thought that you have used 12 ul of enzyme as recommended. I have used frozen tagmented fragments but cannot remember if it was after tagmentation or strand displacement.

Tagmentation enzyme is one of the limiting reagents and you already have used DNA as well. So, there is no harm continuing the protocol. If library yields is not enough then you may prepare another one.
Hi! I just wanted to show my PFGE results. As you and Illumina staff already pointed out, DNA 12000 chip cannot resolve these fragments, so I ran a PFGE and I can clearly see the fragmented DNA. The leftmost lane correspond to the input DNA, the two lanes in the middle different amounts of the same tagmented DNA (4 uL of tagmentation enzyme) and the rightmost lane the 48 kb marker. Thanks everyone for your suggestions.
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Last edited by cerbatana; 07-26-2016 at 01:15 AM.
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