University of Delaware Sequencing & Genotyping Center · University of Delaware Sequencing &...
Transcript of University of Delaware Sequencing & Genotyping Center · University of Delaware Sequencing &...
University of Delaware
Sequencing & Genotyping Center
Erin Bernberg, Bruce Kingham, Olga Shevchenko, Summer Thompson, Mark Shaw
http://www1.udel.edu/dnasequence/Site/Home.html
• 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!
• 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
• 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!
What we like to see
Insect
BacteriaAlgae
Plant
No RNase treatment
What we don’t like to see, but see frequently
Before library prep
After library prep
Too small
3Mb bacterial genome final library profileColumn purified
Size selected to remove 4kb and underAvg. library size ~9kb
Even when everything looks good……
Run metrics
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….
Run metrics
University of Delaware
Sequencing & Genotyping Center
http://www1.udel.edu/dnasequence/Site/Home.html
Plant DNA isolated by an expert
Femtopulse