Molecular Combing Technology:

digital and unbiased quantification of rearrangements 

resulting from targeted genome editing

Alex Simon

April 24th, 2018

NIST-FDA Genome Editing Workshop
The Molecular Combing Technique

Embedding in agarose 

plugs to protect the 

DNA fibers

Extraction of high molecular weight DNA in solution

Combing at a 

constant speed of the 

intact DNA fibers on 

coated coverslip

Direct visualization of 

hundreds of genomes

Cell

harvesting

Enzymatic

treatments and 

washes

 Direct visualization and analysis of single DNA molecules, without amplification

 150-200 human genomes stretched on each coverslip

 Accurate measurement of distances with a constant stretching factor (1μm = 2kbp)
Molecular Combing

FISH/Karyotyping 

Molecular Combing: Range of Use

PCR-based methods

Resolution

500bp 1 Kbp - 1Mbp Chromosomes

Fibers of DNA

Region of Interest

NGS

ddPCR

Designer Nuclease

Detection and Quantitation of Modifications
• Probes: 1 kb to 1000+ kb

• Precision ~3 kb

• Possible multiplexing

Changes in GMC pattern directly indicate 

structural variations with no ambiguity 

The Genomic Morse Code (GMC)

BRCA1 wt

BRCA1 mut

Triplication of 16kbp within the BRCA1 gene – ambiguous detection with CGH & MLPA

triplication 

Sizing of RNU2 array CNV associated to BRCA1 gene

BRCA1 triplication 

HPV16 genome integration (red and blue probes) into human host DNA (green line)
Extraction Hybridization & 

Detection

Image

Acquisition Analysis

1 2 3 4 5

Combing

FiberPrep®

DNA Extraction Kit

FiberProbes®

Genomic Morse Code

FiberVision®

Automated Scanner

FiberStudio®

Analysis Software

FiberComb®

Molecular Combing System

The FiberVision® Molecular Combing Platform

From DNA purification to Data Output 

The platform offers a complete and flexible workflow
U2OS T Cells

96.5% of canonical signal 98.7 % of canonical signal 

High frequency editing with a RNP (RiboNucleoProtein) in U2OS and T cells:

FiberCheck HBB HBD

• Vast majority of signals are canonical, suggesting no large scale rearrangements

• Small percentage of signals are non-canonical

Quantitation of Non-Canonical Signals After Editing*

*Cecilia Cotta-Ramusino et al., CSHL Meeting: Genome Engineering: The CRISPR-Cas9 Revolution, July 21-23, 2017.
Symmetrical:

Same chromosome 

translocations

Loss of Blue Probe:

Single Strand 

Annealing

Others:

Unknown 

Mechanism

Challenging to detect by 

NGS/PCR-based strategy 

due to structural complexity

Challenging to detect by 

NGS/PCR-based strategy 

due to size and unknown 

extent of rearrangement

Not detectable by 

NGS/PCR-based strategy 

due to unexpected nature of 

events

Canonical signal

*Cecilia Cotta-Ramusino et al., CSHL Meeting: Genome Engineering: The CRISPR-Cas9 Revolution, July 21-23, 2017.

Quantitation of Non-Canonical Signals After Editing*
...

Classification Frequency Interpretation

> 150x HG equivalents per slide

10 slides per sample

High frequency editing with RNP in T cells:

Comprehensive detection and quantitation of rearrangements HBB HBD

Single Strand Annealing

Canonical Signal

Same Chr Translocation

Same Chr Translocation

Unknown

Unknown

0.3%

98.7%

0.2%

0.2%

0.4%

0.2%

*Cecilia Cotta-Ramusino et al., CSHL Meeting: Genome Engineering: The CRISPR-Cas9 Revolution, July 21-23, 2017.

Quantitation of Non-Canonical Signals After Editing*
Concluding Remarks

Molecular Combing’s Value for Specificity Measurements:

 Sensitive : >150x coverage per slide; 1500x per sample (~0.25% sensitivity); can be increased

 Digital Quantitation : single-molecule counting of ROI signals

 Unbiased by Complex Patterns & Translocations : visual, direct detection

 Technical Advantages:

• Multiple cell input types

• No amplification bias

• Highly complementary to NGS/PCR based assays

 Currently working with Genome Editing Biopharmas

 Platform ready for translation in Process Development

 Future potential as a QC assay in Manufacturing