university of delaware sequencing & genotyping center · university of delaware sequencing...
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![Page 1: University of Delaware Sequencing & Genotyping Center · University of Delaware Sequencing & Genotyping Center Erin Bernberg, Bruce Kingham, Olga Shevchenko, Summer Thompson, Mark](https://reader035.vdocuments.us/reader035/viewer/2022071118/600d4b68bd356c4f2a39dc17/html5/thumbnails/1.jpg)
University of Delaware
Sequencing & Genotyping Center
Erin Bernberg, Bruce Kingham, Olga Shevchenko, Summer Thompson, Mark Shaw
http://www1.udel.edu/dnasequence/Site/Home.html
![Page 2: University of Delaware Sequencing & Genotyping Center · University of Delaware Sequencing & Genotyping Center Erin Bernberg, Bruce Kingham, Olga Shevchenko, Summer Thompson, Mark](https://reader035.vdocuments.us/reader035/viewer/2022071118/600d4b68bd356c4f2a39dc17/html5/thumbnails/2.jpg)
• PacBio RSII (2011)• PacBio Sequel (2017!)• Illumina HiSeq 2500 (2011)• Illumina MiSeq (2014)• Applied Biosystems 3130xl (2001)
• Advanced Analytical Fragment Analyzer• Advanced Analytical Femtopulse (Coming soon!)• Sage BluePippin• Sage ELF• Covaris S2• Nanodrop (ND100), DeNovix (DS11+), Qubit 3.0
UD is a basic research institution with significant genomics research in Agriculture, Engineering, Biochemistry, Life Sciences, Marine Studies, Environmental Studies.
We are happy to take external and international samples!
![Page 3: University of Delaware Sequencing & Genotyping Center · University of Delaware Sequencing & Genotyping Center Erin Bernberg, Bruce Kingham, Olga Shevchenko, Summer Thompson, Mark](https://reader035.vdocuments.us/reader035/viewer/2022071118/600d4b68bd356c4f2a39dc17/html5/thumbnails/3.jpg)
• Consultation with the potential customer• What is the project? What are your goals?
• Genome size, desired coverage, desired N50, etc.• How are you going to extract your DNA?
• Tissue/source• Individuals, pooled sample
• Method• Yield/ initial QC / how much can you send us?
• QC (all sample tracking is handled through our iLab system)• Nanodrop, Qubit, Fragment Analyzer
• Library Prep• Troubleshooting
• Small run to determine optimal loading concentration• Might be one cell if titration not possible• Troubleshooting
Our workflow
![Page 4: University of Delaware Sequencing & Genotyping Center · University of Delaware Sequencing & Genotyping Center Erin Bernberg, Bruce Kingham, Olga Shevchenko, Summer Thompson, Mark](https://reader035.vdocuments.us/reader035/viewer/2022071118/600d4b68bd356c4f2a39dc17/html5/thumbnails/4.jpg)
• What does HMW DNA mean?
• I extract DNA all the time!
• How hard can it be!
• So you don’t think the NanoDrop is correct?
• But there was 20ug when I gave it to you!
Nothing is more important than the quality of your DNA!
![Page 5: University of Delaware Sequencing & Genotyping Center · University of Delaware Sequencing & Genotyping Center Erin Bernberg, Bruce Kingham, Olga Shevchenko, Summer Thompson, Mark](https://reader035.vdocuments.us/reader035/viewer/2022071118/600d4b68bd356c4f2a39dc17/html5/thumbnails/5.jpg)
What we like to see
Insect
BacteriaAlgae
Plant
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No RNase treatment
What we don’t like to see, but see frequently
Before library prep
After library prep
Too small
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3Mb bacterial genome final library profileColumn purified
Size selected to remove 4kb and underAvg. library size ~9kb
Even when everything looks good……
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Run metrics
![Page 9: University of Delaware Sequencing & Genotyping Center · University of Delaware Sequencing & Genotyping Center Erin Bernberg, Bruce Kingham, Olga Shevchenko, Summer Thompson, Mark](https://reader035.vdocuments.us/reader035/viewer/2022071118/600d4b68bd356c4f2a39dc17/html5/thumbnails/9.jpg)
Try #2: Another extraction using the same kitCoupled with additional DNA cleaning (MoBio)
Size selected to remove 6kb and underAvg. library size ~10kb
Try Again….
![Page 10: University of Delaware Sequencing & Genotyping Center · University of Delaware Sequencing & Genotyping Center Erin Bernberg, Bruce Kingham, Olga Shevchenko, Summer Thompson, Mark](https://reader035.vdocuments.us/reader035/viewer/2022071118/600d4b68bd356c4f2a39dc17/html5/thumbnails/10.jpg)
Run metrics
![Page 11: University of Delaware Sequencing & Genotyping Center · University of Delaware Sequencing & Genotyping Center Erin Bernberg, Bruce Kingham, Olga Shevchenko, Summer Thompson, Mark](https://reader035.vdocuments.us/reader035/viewer/2022071118/600d4b68bd356c4f2a39dc17/html5/thumbnails/11.jpg)
University of Delaware
Sequencing & Genotyping Center
http://www1.udel.edu/dnasequence/Site/Home.html
![Page 12: University of Delaware Sequencing & Genotyping Center · University of Delaware Sequencing & Genotyping Center Erin Bernberg, Bruce Kingham, Olga Shevchenko, Summer Thompson, Mark](https://reader035.vdocuments.us/reader035/viewer/2022071118/600d4b68bd356c4f2a39dc17/html5/thumbnails/12.jpg)
![Page 13: University of Delaware Sequencing & Genotyping Center · University of Delaware Sequencing & Genotyping Center Erin Bernberg, Bruce Kingham, Olga Shevchenko, Summer Thompson, Mark](https://reader035.vdocuments.us/reader035/viewer/2022071118/600d4b68bd356c4f2a39dc17/html5/thumbnails/13.jpg)
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Plant DNA isolated by an expert
Femtopulse