Position (kbp)

Background

Methods

Abstract

(1) Long molecules of DNA are labeled with Bionano reagents by (2) incorporation of fluorophores at a specific sequence motif throughout the genome. (3) The labeled genomic DNA is then linearized in the Saphyr Chip using NanoChannel arrays (4) Single

molecules are imaged by Saphyr and then digitized. (5) Molecules are uniquely identifiable by distinct distribution of sequence motif labels (6) and then assembled by pairwise alignment into de novo genome maps.

Extraction of long DNA moleculesLabel DNA at specific sequence

motifs

Saphyr Chip linearizes DNA in

NanoChannel arrays

Saphyr automates imaging of single

molecules in NanoChannel arrays

Molecules and labels detected in

images by instrument software

Bionano Access software

assembles optical maps

1 2 3 4 5 6

Blood Cell Tissue Microbes

Free DNA Solution DNA in a Microchannel DNA in a Nanochannel

Gaussian Coil Partially Elongated Linearized

©2018 B

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Labeling Human DNA with Bionano’s Direct Labeling Enzyme Avoids Nickase-Based Double-Stranded Breaks and Allows for Chromosome-Arm Length AssembliesHenry B. Sadowski, Zonxiang Zhou, Amy Files, Carly Proskow, Yang Zhang, Michael Saghbini, Alex Hastie, Goran Pljevaljcic, Ernest Lam, Jian Wang,

Luna Zhang, Han Cao and Mark Borodkin

Bionano Genomics, San Diego, California, USA

Bionano genome mapping traditionally images long, megabase sized molecules that are fluorescently labeled using nicking endonucleases

followed by incorporation of fluorescent nucleotides at nick sites. One inherent limitation of nickase-based labeling is the introduction of

systematic double-stranded breaks in regions containing sequence motifs in close proximity on opposite strands.

In order to avoid the inherent limitation of these fragile sites, we have developed an enzymatic direct labeling approach that allows

fluorescent labeling of a specific sequence motif on native double stranded DNA. The direct labeling enzyme shows very high single

molecule sequence specificity and efficiency. By not introducing DNA nicks, direct labeling enables the assembly of genome maps with N50

measurements that now exceed 60 Mbp. DNA labeling and cleanup can be achieved in approximately 5 h with low hands on time.

We present the workflow for direct labeling of ultra-high molecular weight DNA for running on the Saphyr™ system and a comparison of the

results between this new labeling method and the nickase-based labeling. The data obtained using the direct labeling method can be

combined with data from the nickase-based approach in hybrid scaffolding applications for creating high quality genome references. Bionano

genome mapping using this new assay drastically improves SV detection and hybrid scaffolding results all for < $1,000 per sample.

Generating high-quality finished genomes with accurate identification

of structural variation and high completion (minimal gaps) remains

challenging using short read sequencing technologies alone. The

Saphyr™ system provides direct visualization of long DNA molecules

in their native state, bypassing the statistical inference needed to align

paired-end reads with an uncertain insert size distribution. These long

labeled molecules are de novo assembled into physical maps

spanning the entire diploid genome. The platform provides the ability to

correctly position and orient sequence contigs into chromosome-scale

scaffolds and detect a large range of structural variation with very high

efficiency.

ConclusionFragile sites are inherent to nickase-based labeling strategies and limit the assembly of very large genome maps and the sensitivity

to large structural variants. To date, we have leveraged the fact that different nickases will generate different non-overlapping fragile

sites and developed a two nickase NLRS strategy with Nt.BspQI & Nb.BssSI for hybrid scaffolding and SV detection. Nevertheless,

we have continued to explore non-nickase based labeling strategies and have developed the direct labeling and staining chemistry

(DLS) with DLE-1 as the first enzyme in this family. DLE-1 efficiently labels HMW genomic DNA from a variety of organisms in a

highly sequence specific fashion generating very large genome maps and unprecedented assemblies. Direct labeling and staining

(DLS) can now be used for hybrid scaffolding and SV detection with results that exceed those obtained with the two enzyme NLRS

strategy at a cost of <$1000 per sample.

ReferencesMak AC et al. Genome-Wide Structural Variation Detection by

Genome Mapping on NanoChannel Arrays. Genetics. 2016;

202:351-62.

Cao, H., et al., Rapid detection of structural variation in a

human genome using NanoChannel-based genome mapping

technology. Gigascience (2014); 3(1):34

1. Comparison of NLRS and DLS WorkflowsSequence-specific labeling of megabase gDNA for Bionanomapping by Nicking, Labeling, Repairing, and Staining (NLRS).Workflow Overview – 2 days

Sequence-specific labeling of megabase gDNA for Bionanomapping using a Direct Labeling and Staining (DLS)Workflow Overview – 2 days

• DLS labeled NA 12878 DNA run on Saphyr system to 70x coverage

• Molecules → Haplotype Aware Assembly → Genome Maps

• Two assembled genome maps spanning the MHC locus (Hap1 & Hap2)

• Ref 6= PGF (Ref sequence for chr 16 MHC region)

• Empty area= no DLE-1 sites, Blue= aligned labels, Black= nonaligned labels

7. DLS Assembly across the human Chr6 MHC locus

450kb365kb

285kb225kb

825kb785kb

680kb610kb

Fresh Blood Plug Lysis DNA

60

0n

g native

DN

A

75

0n

g native

DN

A

60

0n

g Nt.B

spQ

INLR

S

75

0n

g DLS (-) d

ye re

mo

val

DLS (+) d

ye re

mo

val

2. PFGE Results Comparing NLRS and DLS

SampleMolecule N50 > 150 Kbp

(Kbp)

Bionano Map N50

(Mbp)

NA12878 293 55.9

Human Fresh Blood 307 56.9

Bionano Maize B73 260 100.0

Durum Wheat 364 13.0

Farro 300 32.7

Strawberry 241 13.3

Kakapo 247 69.3

Hummingbird 310 38.7

Blackbird 243 21.6

Fish 245 22.3

Ferret 262 66.1

Rat 251 Pending

Pig 335 65.2

Soybean 246 23.0

Brassica 270 12.4

Mouse 280 101.4

6. De novo Assembly of Diverse Genomes with DLS • Enables much larger genome maps

• Achieving molecule length N50s >300kbp for good samples regularly

• Molecule breaks are random

• Molecules >2Mbp were observed

• A 2.34Mbp molecule aligning to human chromosome 3 is shown below

Molecule to Reference Alignment

3. DLS Molecules are Long

2.34Mbp molecule

BspQI

Ref

Diploid

Maps

Ref

Map -

Hap1

Map -

Hap2

Single

Molecules

DLE-1

5. Full Chromosome Arms of human Chr3 assembled with DLS

Molecule

Reference

Molecules

• DLE-1 is the first of a new class of Bionano DLS enzymes

• Single enzymatic reaction; no nicking; no repair step; not NLRS

• No fragile sites…optical maps are ~60x higher (human)

• Works on the same principle of tagging recognition motifs; highly

specific

• Long molecules + No Fragile Sites + Increased Label Density

4. DLS (Direct Label and Stain) Labeling Chemistry

• Above charts are for heterozygous SV calling; homozygous calling is more sensitive

• DLE-1 enables unmatched sensitivity to large SV’s at PPV’s close to 100%

• Sensitive to insertions and deletions as low as 500bp

8. In silico SV Detection with Bionano Maps (human)

Hap1

Hap2