Failed tagmentation reaction for Nextera library?

[sweetph3]

Just had failures #2 and #3 using Illumina Nextera. I was able to run a sample from failure #1 of the post-zymo cleanup on Agilent 2100 Bioanalyzer this morning, and it looked almost identical to the input genomic DNA. Haven't run the samples from todays failures, but suspect they would look similar. This points to a failed tagmentation reaction. Anyone else ever experience this? If so, is it due to bad enzyme, or a problem with your input DNA? FYI, the 3 samples came from 2 different labs/ 2 different species. #2 was a re-precipitate of #1 to remove any potential inhibitors present in the resuspension buffer. I'd really appreciate any input you might have!

[ZWB]

I've had similar problems in the past with failed tagmentation and it was always due to contaminants/inhibitors in the sample. In my experience, the MoBio power clean kit seems to do the best job removing inhibitors.

[sweetph3]

I finally had a chance to run 3 different clean-up protocols and make Nextera with the resulting DNA. I used Qiagen plant DNeasy miniprep, MoBio Power Clean DNA Clean-Up Kit, and Zymo Research OneStep PCR Inhibitor Removal Kit. The only one that gave a result was the MoBio Power Clean. I finally had a detectable (by Qubit) concentration of DNA in my library, and on the Bioanalyzer I finally see fragments 300bp to >1kb. All others I still only see some remnant genomic input on the Bioanalyzer. THANK YOU to ZWB for your suggestion, now I can move forward with this library after a month of pulling out my hair and many apologies and explanations to our collaborator. Hooray!

[Sully]

I am having similar troubles at the tagmentation step and I hope someone can make comments on this; I have checked the quality of my gDNA samples on gel, obtained 260/280 and 260/230 ratios on nanodrop and quantified them using Qubit, cross checked the concentration with QuanIT standard curve, also. But I consistently get bioanalyzer profile with the peak centered around 1.2 kb under standard conditions (5 min, 55 C). Interestingly, protracted incubation up to ten min produced very similar profiles, too. I then started with 25 ng total gDNA, which improved the profile but the peak is sitting around 1 kb. I have also included control DNA (lambda DNA which comes with another commercial kit and quantified by the manufacturer) and obtained the same exact profile. My thermocyler and pipettes are calibrated annually. I am almost ready to conclude the nextera enzyme I have received is from a bad lot. What else I may try before contacting Illumina? Thank you.

[Simone78]

I am having similar troubles at the tagmentation step and I hope someone can make comments on this; I have checked the quality of my gDNA samples on gel, obtained 260/280 and 260/230 ratios on nanodrop and quantified them using Qubit, cross checked the concentration with QuanIT standard curve, also. But I consistently get bioanalyzer profile with the peak centered around 1.2 kb under standard conditions (5 min, 55 C). Interestingly, protracted incubation up to ten min produced very similar profiles, too. I then started with 25 ng total gDNA, which improved the profile but the peak is sitting around 1 kb. I have also included control DNA (lambda DNA which comes with another commercial kit and quantified by the manufacturer) and obtained the same exact profile. My thermocyler and pipettes are calibrated annually. I am almost ready to conclude the nextera enzyme I have received is from a bad lot. What else I may try before contacting Illumina? Thank you.

it could be that the enzyme comes froma bad lot but why using so much for tagmentation? 25 ng is not necessary! Reducing the input will help to give shorter libraries and the data quality won´t be affected. Prolonged incubation at 55 degrees, like it was reported in the previous post, doesn´t work because when the transposase ligates adaptors it becomes inactive. So, if after 5 min there is no more transposes loaded with oligos, even incubating 1 hour won´t change the size distribution of the library.

[mdalessio]

Has someone already optimized the input DNA for genomic libraries, or is this something everyone should optimize themselves?

[lbcbci]

Nextera DNA sample kit protocol

I am recently using Nextera DNA sample kit for library prep. According user guide, the spin speed was set as 1300xg for PCR clean up with Zymo column. Actually there is only about 4000rpm (Eppendorff centrifuge) and the column looks wet still after spin. Is anybody aware about this? Does it better to increase spin speed?

Appreciate your sharing!

LBC

[Simone78]

if you read the manual from Zymo, they usually recommend >10,000g. I don´t understand why Illumina says one should use a (much) lower speed. I do all my spins at 10,000 and the columns is dry afterwards. Don´t worry, you´re not going to destroy the filter!

[lbcbci]

Thanks a lot, Simone 78.
It is highly possible illumina's typo problem since 13,000g would be very close to 11,000rpm which is typical number for the community.
But I just curious dose anybody have any problem when strictly follow the protocol at 1,300g? There should be a lot of kits have been run by now.

[lac302]

Struggling with some fungal samples using Nextera XT. I use this kit routinely samples ranging from arabidopsis to salmonella. gDNA is extremely clean based on Nanodrop/PicoGreen and agarose gel. Not getting enough detectable PCR product to warrant even moving forward with bead-based normalization.

It's not a kit issue since I've run through several lots at this point...have had success with bacterial libraries in the same time frame.

I typically do the extractions myself but the gDNA coming to me has been made using a variety of methods. The last prep being phenol, phenol/chlroform extracted, ethanol precipitated and run over a column.

Really stumped here. I'd like to figure out with the inhibitor is but will be moving on to mechanical fragmentation at this point.

Is the enzyme the same between Nextera and Nextera XT? Has anyone resolved similar issues?

[nucacidhunter]

I think the only difference is concentration of enzymes. Following to consider:
1- The buffer used to elute from columns. If TE has been used, inhibition of tagmentation enzymes is expected.
2- If you have enough DNA you can do another clean up with 1.2x AMPure bead or a column and elute in TE0.1
3- GC content of genome, normally %GC 30-75 is OK

[sweetph3]

Any time I've had issues, especially if the gDNA was prepared by another group, I start off with the MoBio Power Clean now. It hasn't failed me yet. I think some samples have inhibitors that carry over even through columns. Good luck!

[lac302]

I think the only difference is concentration of enzymes. Following to consider:
1- The buffer used to elute from columns. If TE has been used, inhibition of tagmentation enzymes is expected.
2- If you have enough DNA you can do another clean up with 1.2x AMPure bead or a column and elute in TE0.1
3- GC content of genome, normally %GC 30-75 is OK

I think Qiagen EB buffer was used. Either way these samples are fairly concentrated. I end up diluting close to 500x in water. GC content is around 48%, so no issues there.

Any time I've had issues, especially if the gDNA was prepared by another group, I start off with the MoBio Power Clean now. It hasn't failed me yet. I think some samples have inhibitors that carry over even through columns. Good luck!

It's definitely an inhibitor issue. Possibly carbohydrates...We have powersoil kits on hand that I'm trying today.

[nucacidhunter]

I wonder if you could post DNA recovery using MoBio kit if you are measuring it. Zymo also has an inhibitor removal kit (OneStep™ PCR Inhibitor Removal Kit) whit simpler workflow than MoBio that claims 50-90% recovery.

[cerbatana]

EDIT:

I just realized that the original post was about Nextera kit while the one I' using is Nextera mate-pair kit. I'll create a new thread so this post can be removed. Thank you.

Hi! I'm also having problems with the same 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.

What happened?