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

Page 3

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

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

0

60 min

DNA Buffer

9

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