fresh blood plug lysis dna bspqi - bionano genomics€¦ · combined with data from the...
TRANSCRIPT
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