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Old 03-31-2016, 07:06 PM   #1
user 31888
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Default Truseq Exome Library Prep: amplification does not work

Hi,

I have 6 samples with which I am trying to make a library with Illumina TruSeq Exome Library Prep Kit (starting material was 100-300 ng).

The problem is that before the exome capture, the first enrichment of my fragments does not work (fragment concentrations are in a 0.5 - 3 ng/ul range measured with PicoGreen).
Knowing that I need to pull 200 ng of fragments per sample before exome capture, I cannot even go farther.

I used these same samples to do qPCRs (KAPA) and whole genome amplifications (QIAGEN Repli-g) and it worked great.

Illumina tech support has not been very helpful so far, so I would have few questions:
1) Do you think I could use the QIAGEN Whole Genome Amplification kit I used before instead of Illumina reagents?
2) Any impact on the adaptor sequences? (I don't know what amplification method is used in the Illumina kit)
3) Does this happened to someone before? Can the kit reagents be faulty?

Thanks !
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Old 03-31-2016, 08:56 PM   #2
nucacidhunter
Jafar Jabbari
 
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This indicates sub-optimal library prep. You can use WGA on prepared libraries to increase yield but your amplified library will not have correct structure and mostly will consist of mass of DNA without flanking Illumina Adapters which are not useful for hybridisation and capture.
Illumina uses reverse and forward primers for PCR enrichment.
Kit reagents can be faulty, but one cannot rule out input DNA and library prep reaction set up either without further work.
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Old 04-05-2016, 01:58 PM   #3
bilyl
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Quote:
Originally Posted by user 31888 View Post
Hi,

I have 6 samples with which I am trying to make a library with Illumina TruSeq Exome Library Prep Kit (starting material was 100-300 ng).

The problem is that before the exome capture, the first enrichment of my fragments does not work (fragment concentrations are in a 0.5 - 3 ng/ul range measured with PicoGreen).
Knowing that I need to pull 200 ng of fragments per sample before exome capture, I cannot even go farther.

I used these same samples to do qPCRs (KAPA) and whole genome amplifications (QIAGEN Repli-g) and it worked great.

Illumina tech support has not been very helpful so far, so I would have few questions:
1) Do you think I could use the QIAGEN Whole Genome Amplification kit I used before instead of Illumina reagents?
2) Any impact on the adaptor sequences? (I don't know what amplification method is used in the Illumina kit)
3) Does this happened to someone before? Can the kit reagents be faulty?

Thanks !
What is your total elution volume? What kind of total ng output are you getting from the first round of PCR? How many cycles of PCR are you using? Are you doing size selection? What is your shear size?

It might be more prudent to use the Kapa Hyper Prep kit for the library prep process (with the TruSeq Exome adapters, of course), followed by Kapa HiFi for the PCR step. You'll get much higher yields.
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