FEMTO Pulse® QC Checkpoints Optimize and Accelerate Large ... · Peak Size Conc. From To Avg. Size...
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FEMTO Pulse® QC Checkpoints Optimize and Accelerate Large Insert SMRTbell® Library Preparations for PacBio
Transcript of FEMTO Pulse® QC Checkpoints Optimize and Accelerate Large ... · Peak Size Conc. From To Avg. Size...
FEMTO Pulse® QC Checkpoints Optimize and
Accelerate Large Insert SMRTbell® Library
Preparations for PacBio
University of Delaware
Sequencing & Genotyping Center
Bruce Kingham, Olga Shevchenko, Erin Bernberg, Mark Shaw, Salma Kaouser
[email protected] http://www.Udel.Edu/DNA
Extraction of HMW DNA
merges science and art
The importance of HMW DNA
for resolving complex genome
structures using PacBio data
• High quality genomic DNA
– NEED: High MW DNA to make a large fragment library
– IMPORTANT: Need a good extraction method, procedure and operator.
• A robust shearing method
– NEED: To reproducibly shear DNA to the desired level
– IMPORTANT: Complications increase time, sample use, costs
• High quality QC analysis
– NEED: Way to accurately measure & quantify the DNA integrity & size
– IMPORTANT: Failure to qualify increases sample use, time, costs
– Positive impact on results
Effective Library Construction Requires
FEMTO Pulse™• Built on the Fragment Analyzer frame
– Holds a 12 capillary array
– 3 x 96 well plates
– Two gel bottles
• Significant and innovative changes
• Only pulsed-field capillary electrophoresis instrument
on the market
– Automate your analysis – save time & reduce effort
• Separate DNA through 200,000bp in ~1 hour
• Femtogram level sensitivity (in well concentration)
Visualize Actual Genomic DNA Distributions
• The pulsed power supply gives a true separation of genomic
DNA
– Eliminates compression of HMW fragments
– Improves separations
– Enhances resolution of large DNA fragments
– Tailor methods - different methods for different analyses
– Only needs picograms starting amounts of DNA
• There is no large fragment stacking as found in
constant field electrophoresis
Fragment Analyzer™
FEMTO Pulse™
Average Size
Black = 37,396bp
Blue = 69,257bp
Average Size
Black = 59,023bp
Blue = 79,535bp
gDNA 165Kb Analysis Kit
gDNA 50Kb Analysis Kit
Sample: 1 BP 1:160 1ul
Well Location: G2
Created: 04-Sep-18 12:42 PM
Peak Size Conc. From To Avg. Size CV% RFU Corr. Peak Area
Sample: 1 Qubit 1:1600 4 ul
Well Location: G6
Created: 04-Sep-18 12:42 PM
Peak Size Conc. From To Avg. Size CV% RFU Corr. Peak Area
Sample run on FEMTO Pulse
Same sample, but this is the dilution
measured on the Qubit, than directly
run on the FEMTO Pulse
Completed SMRTbell library – 10Kbp Blue Pippin cut
Chlorovirus DNA, genome size ~350-500Kbp,
is presumably the larger fragment.
FEMTO Pulse allowed us to resolve what
may potentially be unexpected virophage DNA.
We are currently preparing these samples
for sequencing
Sample: A6 gDNA 1:400 1ul
Well Location: E11
Created: Wednesday, February 28, 2018 11:53:51 AM
Run metrics for a library prepped using Fragment Analyzer data
Sample: A6gdna qubit 1ul
Well Location: G6
Created: 30-Mar-18 3:32 PM
Peak Size Conc. From To Avg. Size CV% RFU Corr. Peak Area
Improved metrics, same library re-prepped using FEMTO Pulse data
FEMTO Pulse™
• Decrease analysis time
– Reduce time from 16 hours
to about 1 hour
• Be more efficient
– Eliminate manual steps and
inefficiencies
• Increase productivity
– Less hands on time and
more automation
• Use less input material
– Retain precious samples for
real analysis
• Worry free sample loading
– Eliminate concentration
dependent sizing artifacts
• Reduce labor
– Save time
• Better data
• Electropherograms provide
more details, easier to see small differences
• Real data analysis
• No peak compression artifact
• Precise HMW sizing
Product Note
Workflow Solutions
Fast, High-Resolution DNA Sizing with the FEMTO Pulse System
Accurately size high molecular weight DNA smears up to 165 kb in
under 1.5 hours for generating large-insert SMRTbell libraries
PFGE FEMTO Pulse
Size Resolution up to 10 Mb up to 165 kb
Run Time up to 20 Hr <1.5 Hr
DNA Input >50 ng >10 ng
Gel Staining Yes No
Accuracy High High
Quantification Not automated Automated
www.pacb.com/wp-content/uploads/Product-Note-Fast-High-Resolution-DNA-Sizing-with-the-FEMTO-Pulse-System.pdf
University of Delaware
Sequencing & Genotyping Center
Bruce Kingham, Olga Shevchenko, Erin Bernberg, Mark Shaw, Salma Kaouser
http://www.Udel.Edu/DNA
