University of Delaware Sequencing & Genotyping Center · University of Delaware Sequencing & Genotyping Center Erin Bernberg, Bruce Kingham, Olga Shevchenko, Summer Thompson, Mark

University of Delaware

Sequencing & Genotyping Center

Erin Bernberg, Bruce Kingham, Olga Shevchenko, Summer Thompson, Mark Shaw

[email protected]

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

[email protected]

http://www1.udel.edu/dnasequence/Site/Home.html

Plant DNA isolated by an expert

Femtopulse