Sorting out the Good from the Great ~ Sample QC · 2016. 9. 4. · Sorting out the Good from the...
Transcript of Sorting out the Good from the Great ~ Sample QC · 2016. 9. 4. · Sorting out the Good from the...
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Sorting out the Good from the Great ~ Sample QC ~
Melissa Huang Liu, Ph.D.
Product Manager, 2100
Bioanalyzer System &
Applications
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Outline
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Importance of Quality Control
2100 Bioanalyzer & 2200 TapeStation Systems
Sample QC in Genome Editing
Sample QC in Functional Genomics
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Outline
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Importance of Quality Control
2100 Bioanalyzer & 2200 TapeStation Systems
Sample QC in Genome Editing
Sample QC in Functional Genomics
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Quality of Samples is Critical for Experimental Success
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Reliability of results
Reducing sample repeats (saving cost & time)
Reproducibility of results from various sites and operators
Avoiding exponential increase of data analysis effort
Garbage in, garbage out!
QC of starting material and after critical workflow steps
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Outline
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Importance of Quality Control
2100 Bioanalyzer & 2200 TapeStation Systems
Sample QC in Genome Editing
Sample QC in Functional Genomics
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Electrophoresis Product Portfolio
2100 Bioanalyzer System –
Electrophoresis in microchannels
• Analysis of biomolecules: DNA, RNA, Proteins
and Cells
• RIN for user-independent RNA QC
2200 TapeStation System -
ScreenTape Technology
• fully scalable
• 96-well plate compatible
• RINe for user-independent RNA QC
• DIN for user-independednt gDNA QC
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Assay Portfolio
DNA1000 DNA7500 DNA12000 High
Sensitivity
DNA
6000
Nano
6000 Pico Small
RNA
P230 P80 HSP 250
Flow
Cytometry
DNA assays
RNA assays
Protein assays
Flow Cytometry
Assay
HS D1000 D1000 Genomic DNA
RNA HS RNA
P200
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• Sizing, Quantitation
• gDNA integrity (DIN)
• PCR products, digests, larger DNA fragments
• Sizing, Quantitation
• total RNA, mRNA
• Purity & integrity determination (RIN; RINe)
• Sizing, Quantitation
• cell lysates, column fractions, purified proteins,
antibodies etc
• Two color detection
• Analysis of protein expression in cells
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Slab gel analysis - a bottleneck in Protein & DNA analysis
Manual process
Difficult to automate
Slow
Not accurate enough
Bad reproducibility
No direct comparison
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Automated Electrophoresis has the potential to address this
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Overview run workflow
1 = ~12 samples
in 30-40 minutes
1 = 2-16 samples
in 10-20 minutes
Prepare gel/dye mix
Prime Chip
Prepare Chip
Select assay
Run
Prepare samples
Load Tips and Samples
Automatic Assay selection
Run
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60 min
DNA Buffer
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0 40 min
0 20 min
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Outline
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Importance of Quality Control
2100 Bioanalyzer & 2200 TapeStation Systems
Sample QC in Genome Editing
Sample QC in Functional Genomics
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DNA& RNA Applications in Genome Editing
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Sample QC
in NGS
gDNA
integrity
PCR
products
Restriction
Digest
Analysis
Total RNA
integrity
oligo
QC
Oligo tailing reaction
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Example of QC steps in a complex workflow: NGS
Adapted from Quail et al (2008) Nature Methods)
Integrity of starting material
Size distribution after fragmentation
Size distribution of adapter-ligated fragment library
Quality control and quantification after PCR
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Examples of sample QC steps in SynBio workflows Vector Assembly
Starting material QC: Gene of interest after PCR
amplification
Verifying vector composition with restriction analysis
Vector analysis by NGS (including multiple QC steps)
CRISPR/ Cas9
DNA target after PCR amplification
Cleavage efficiency of digestion with Cas9 Nuclease
guide RNA synthesis
Starting material QC: oligo templates
Monitoring oligo annealing reactions
quantification of gRNA product
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Outline
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Importance of Quality Control
2100 Bioanalyzer & 2200 TapeStation Systems
Sample QC in Genome Editing
Sample QC in Functional Genomics
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RNA Applications in Functional Genomics
Gene
Expression Transcriptome
Profiling
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RNA QC for
qPCR
RNA QC for
microarrays RNA QC for
NGS
smallRNA
QC
mRNA
extraction
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Protein Applications in Functional Genomics
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Monitor
Protein
Purification
Protein
Expression
in
cell lysates
Protein QC
prior to MS
analysis
Protein
quantitation
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More Information
www.agilent.com/genomics/bioanalyzer
www.agilent.com/genomics/tapestation
• Application Notes
• Publications
• Data Sheets
• Videos
• Brochures
• Special Offers
• Free Software Downloads
www.agilent.com/genomics/sample_QC
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http://www.agilent.com/genomics/bioanalyzerhttp://www.agilent.com/genomics/tapestationhttp://www.agilent.com/genomics/sample_QC