Valérie Reeb and Erik TwaitState Hygienic Laboratory at the University of Iowa
GenomeTrakr meetingArlington, VA
September 26-28, 2018
Adjustable Tip Spacing Pipettes, an Alternative to Automated Library Prep
Automated Genomic DNA isolation
High qualityDNA Isolated
Gram+ protocol
QIAamp DNA Mini kit
24 isolates on 2 QIAcubes~3.5 hours
Enzymatic digestion with lysozyme and RNase A
Automated Liquid Handlers for Library preparation
Illumina qualified instruments for Nextera XT DNA library prep- Beckman Coulter Biomek iSeries- Eppendorf epMotion 5075t- Tecan Freedom EVO NGS- Hamilton Robotics NGS STAR
Cons- Instrument cost: $100,000 to $200,000- Service contract: $9,000 to $19,000/year- Increased cost for reagents and plastics- Instrument failure if only 1 instrument- Not practical for low sample volume
Pros- Reduced hands-on time- Minimize errors- Enables higher throughput
Automated Liquid Handlers for Library preparation
E1-ClipTip EqualizerThermo Fisher Scientific
E4 XLSRainin
Voyager IIIntegra
Adjustable Tip Spacing Pipettes
Advantage of adjustable tip spacing pipettes
- Transfer of samples between labware formats- Low cost: ~ $1,800 – $2,600 / pipette
6 pipettes ~ $9,000
- Reduce technician hands-on time compare to using regular single and multichannel pipettes
3h from gDNA quantification to libraries quantification (16 libraries)
- Minimal repetitive stress- Reduces potential pipetting errors- Increase quality consistency across library preparation
Ideal for our public health laboratories with medium throughput sequencing
Various models available • channels: 4 - 12• volume: 0.5 -1250 µl• tip spacing : 4.5 mm and 33 mm• well plates: 12 - 384
Touch wheel interface • one-handed programming• menu navigation• on the fly changing of tip spacing
Integra Voyager II Series Pipettes
8 channels12.5 ul (0.5 – 12.5 ul); 4.5 – 14 mm
300 ul (10 – 300 ul); 9 – 14 mm
Program Function
QUBIT DNA extracts or libraries are transferred from 1.5 ml tubes to 0.5 ml Qubit tubes for quantification
DNA DILUTE 2X DNA extracts are transferred from racked 1.5 ml tubes to a 96-well plate and diluted to 0.2 ng/µl
TAGMENT Tagmentation reactions containing ATM, TD buffer and DNA extracts are mixed before placing in the thermal cycler
NT STOP NT buffer is added and the reactions are mixed, stopping tagmentation
PCR PCR reactions containing NPM, index adapters and tagmented gDNA are mixed
DNA-BEADS MIX DNA libraries and AMPure XP beads are mixed in a 96-well plate
BEAD WASTE Buffer is removed from the DNA/AMPure XP bead complexes and transferred to a waste reservoir
POST-AMP WASH DNA/AMPure XP bead complexes are washed twice with 80% ethanol
RSB MIX DNA/AMPure XP bead complexes are mixed with RSB buffer
LIB TRANSFER Eluted DNA libraries are transferred from a 96-well microplate to racked 1.5 ml microfuge tubes
Custom DNA Library Prep Programs
Genomic DNA transfer to dilution plate
1.5 ml tubes -13 mm spacing 96 well PCR plate - 9 mm spacing
Cleaned libraries transferred to tubes
Midi wash plate - 9 mm spacing 1.5 ml tubes - 13 mm spacing
Funding Epidemiology and Laboratory Capacity for Infectious Diseases grant, CDC
SHL genomic teamErik TwaitValérie Reeb, Ph.D.Jeff BenferLucy DesJardin, Ph.D.Gina KlineAlex TrannelStephany CochranRyan Jepson
Acknowledgments
Thank you
Mission: The State Hygienic Laboratory at the University of Iowaprotects and improves quality of life by providing reliableenvironmental and public health information through thecollective knowledge and capabilities of our organization.
Valérie [email protected]
