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Old 11-13-2014, 08:30 AM   #1
cybeline
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Default MDA+Nextera?

Hello,

I am dealing with problematic soil environmental samples with low HMW DNA and low yield. I have tried to make metagenomic libraries using Nextera XT kit but without luck.

What some other people have done in the field is to use whole genome amplification by multiple displacement amplification (MDA) to get enough DNA and then sequence it using 454.

What I was wondering is, could I use use Nextera library prep (input DNA 50ng) (not Nextera XT) instead of TruSeq library prep for Illumina on MDA amplified samples.

Has anyone seen/done/heard for MDA samples + Nextera (not in single cells).

While I am concerned with using only 50ng of input DNA when I get ug amounts from the MDA, we would be doing replication to capture more diversity of the sample.

Thank you.
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Old 11-13-2014, 11:55 PM   #2
nucacidhunter
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MDA introduces lots of artefacts and I would consider it as a last resort. I have seen manufacturers results with E. coli DNA having only 70% of reads mapping to reference after MDA. Nextera XT does not require HMW input DNA. I would consider cleaning DNA with one of commercially available spin columns designed to remove inhibitors. Nextera is very sensitive to contaminants and consistently performs well with clean DNA as recommended by Illumina.
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Old 11-17-2014, 05:03 AM   #3
cybeline
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Quote:
Originally Posted by nucacidhunter View Post
MDA introduces lots of artefacts and I would consider it as a last resort. I have seen manufacturers results with E. coli DNA having only 70% of reads mapping to reference after MDA. Nextera XT does not require HMW input DNA. I would consider cleaning DNA with one of commercially available spin columns designed to remove inhibitors. Nextera is very sensitive to contaminants and consistently performs well with clean DNA as recommended by Illumina.
Thank you very much.
Yes, I am aware of all the bias introduced by MDA and I have also tried to clean my DNA using spin columns (Zymo genomic DNA kit and Qiagen dna clean) but I seem to lose most of the little DNA that I have from it and then still not able to make the Nextera XT libraries.
I am thinking that it may be a combination of little DNA and contaminants.
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Old 11-17-2014, 05:24 PM   #4
nucacidhunter
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I have had good results by cleaning up with zymo gDNA clean and concentrater. One option to increase yield would be to elute with warm (55-60 C) Tris pH8 (Qiagen EB buffer) and incubate at the same temperature for 5 mins. You can also do a similar second elution and that would give another 10-15% of 1st elution yield. Generally, I only loose 10-15% with these columns.

You can try MDA as well but it is not supported. The issue with MDA would be branched nature of product and you may need to optimise protocol.

Last edited by nucacidhunter; 11-17-2014 at 05:29 PM.
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Old 11-25-2014, 05:59 AM   #5
sweetph3
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I had run into issues with inhibitors in DNA preps when trying to make Nextera libraries. The only kit that removed the inhibitors was MoBio PowerClean DNA kit. Zymo and Qiagen didn't work. MoBio also makes a soil DNA extraction kit that I'm told by colleagues is excellent. How are you isolating your DNA? Perhaps this would work for you. (No, I don't work for MoBio, I've just been very impressed by their kits.)
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Old 03-01-2018, 03:11 AM   #6
JBKri
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Quote:
Originally Posted by cybeline View Post
Hello,
...
What I was wondering is, could I use use Nextera library prep (input DNA 50ng) (not Nextera XT) instead of TruSeq library prep for Illumina on MDA amplified samples.
...
Thank you.
I am facing a similar choice. I have no previous experience with MDA, but my thought is to use the Nextera XT kit and thus avoid the MDA. I have the impression that biases from MDA are far worse than from the XT PCR step. Thoughts?
Jon
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Old 03-01-2018, 08:07 PM   #7
nucacidhunter
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It depends on the required coverage. If up to 100M reads will give enough coverage then Nextera XT is better options. MDA normally will result in fusions which could be detrimental to your experiment.
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Old 03-01-2018, 08:29 PM   #8
SNPsaurus
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We've had some issues with different WGA libraries and Nextera in that WGA methods often create lots of ssDNA which will interfere with proper quantification and perhaps sop up transposomes.
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