Optical mapping was run on the Saphyr platform6 at Bionano Genomics (San Diego, CA, United States). The optical maps were analyzed using the Bionano-solve pipeline7 . Briefly, the maps were detected using AutoDetect, and assembled using the de novo assembly package AssembleMolecules. The resulting consensus maps were aligned to Hg19/GRCh37 using the Bionano RefAligner. Lastly, the variants of interest were visualized using Bionano Access, and the resulting smap files were converted to VCF using a custom version of the smap2vcf script8 . De novo SVs were discovered by merging these VCF files into a single trio-VCF. The SVs were merged using SVDB v2.3.0, and variants unique to the proband were discovered using the GNU grep tool (Eisfeldt et al., 2017 (link)).
Saphyr platform
Manufactured by Bionano Genomics
Sourced in United States
The Saphyr platform is a laboratory instrument designed for genome analysis. It utilizes optical mapping technology to detect large structural variations in DNA samples. The Saphyr platform provides high-resolution, long-range genomic information to researchers and clinicians.
Automatically generated - may contain errors
Lab products found in correlation
Refaligner, by Bionano Genomics (3 mentions)
Solve pipeline, by Bionano Genomics (1 mentions)
Bionano solve, by Bionano Genomics (1 mentions)
Access software, by Bionano Genomics (1 mentions)
Ix71 microscope, by Olympus (1 mentions)
Nanobind disk, by Bionano Genomics (1 mentions)
5.5 scmos camera, by Olympus (1 mentions)
7 protocols using saphyr platform
1
Optical Mapping for Structural Variant Detection
2
Optical Mapping of Genomic DNA Using Saphyr
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Optical mapping was performed on genomic DNA from the proband by running dual enzymes (BspQI, BssSI) on the Bionano Genomics (San Diego, CA, USA) Saphyr platform (https://bionanogenomics.com/support-page/saphyr-system ). Analysis was performed as described previously (Eisfeldt et al. 2019 (link)). Briefly, the optical maps were analyzed using Bionano-solve (Bionano-solve">https://bionanogenomics.com/support-page/Bionano-solve ), aligned to Hg19 reference genome using Bionano RefAligner (version 5649) and output files were converted into VCF files using a custom script (https://github.com/J35P312/smap2vcf ). Variants of interest were visualized in Bionano access.
3
Bionano Plant DNA Extraction
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Flash-frozen leaf tissue (see above) was shipped on dry ice to the McDonnell Genome Institute at Washington University in St. Louis. DNA was isolated with the Bionano plant tissue hybrid protocol (liquid N2 grinding and tissue ruptor), including density gradient purification of nuclei, which were then embedded in agarose plugs prior to DNA extraction. Labeling was performed with a Bionano DLS Kit followed by analysis on a Bionano Saphyr platform, generating an estimated genome coverage of 214×. Computational analysis was performed with Bionano Access software.
4
Genomic Profiling of Eμ-Ret B-ALL
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Genomic DNA from 18 Eμ-Ret B-ALL and 6 transgene-negative BALB/c control B cell samples was analyzed by chromosome microarray (CMA) at the Fox Chase Cancer Center Genomics facility. CEL file output from the Affymetrix Mouse Diversity Genotyping Array was analyzed using the Golden Helix SNP and Variation Suite (SVS) analysis software (Bozeman, MT). Intensity data was interpreted directly from the raw file source to call and characterize copy number variants (CNV) in each sample, and resulting visualizations were generated using the standard workflows in the Golden Helix software package. For optical genome mapping (OGM), high molecular weight DNA from 3 additional Eμ-Ret leukemia samples was analyzed on the Saphyr platform by Bionano Genomics (San Diego, CA), using their rare variant analysis workflow. Data for structural variation detection was processed using Bionano Access software.
5
Nanochannels-based DNA Sizing in Saphyr
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The
DNA extracted in the device (sans heat
concentration) and using the Nanobind disk were sized in a research-grade
version of the Saphyr platform developed by Bionano Genomics Inc.26 (link) The DNA from either extraction method was suspended
in 1× Bionano flow buffer along with dithiothreitol at a concentration
of 0.1 M and YOYO-1 dsDNA stain at a concentration of 0.5 μM.
For each sizing experiment, 8 and 11 μL of this DNA mix was
loaded in the reservoirs labeled as inlet and outlet on the Saphyr
flow cell, respectively. The DNA were electrokinetically loaded into
the roughly 34 nm wide and 1 mm long square nanochannels using a custom
electrophoresis script.27 (link) The fluorescently
labeled backbone of the stretched DNA molecules was illuminated using
an OBIS (488 nm, 150 mW, Coherent) laser and imaged using an Andor
Zyla 5.5 sCMOS camera with a 60× air objective (NA = 0.90), mounted
on an Olympus IX-71 microscope. The images were processed using the
Bionano image processing algorithm (available from Bionano Genomics),
and the DNA sizes calculated using a 1 pixel = 366 bp conversion factor.
DNA extracted in the device (sans heat
concentration) and using the Nanobind disk were sized in a research-grade
version of the Saphyr platform developed by Bionano Genomics Inc.26 (link) The DNA from either extraction method was suspended
in 1× Bionano flow buffer along with dithiothreitol at a concentration
of 0.1 M and YOYO-1 dsDNA stain at a concentration of 0.5 μM.
For each sizing experiment, 8 and 11 μL of this DNA mix was
loaded in the reservoirs labeled as inlet and outlet on the Saphyr
flow cell, respectively. The DNA were electrokinetically loaded into
the roughly 34 nm wide and 1 mm long square nanochannels using a custom
electrophoresis script.27 (link) The fluorescently
labeled backbone of the stretched DNA molecules was illuminated using
an OBIS (488 nm, 150 mW, Coherent) laser and imaged using an Andor
Zyla 5.5 sCMOS camera with a 60× air objective (NA = 0.90), mounted
on an Olympus IX-71 microscope. The images were processed using the
Bionano image processing algorithm (available from Bionano Genomics),
and the DNA sizes calculated using a 1 pixel = 366 bp conversion factor.
6
Quantifying 45S rDNA Arrays in Barley Optical Maps
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Regular arrays of 45S rDNA units can be recognized and quantified in optical maps generated on the Saphyr platform (Bionano Genomics) thanks to the presence of a DLE‐1 labelled site in the 26S rRNA gene, which generates a regular pattern with ~9–10 kb label spacing (Figure 6a ). The units were quantified from size‐filtered (>150 kb) raw (single‐molecule) data of DLE‐1 optical map of barley cv. Morex (Mascher et al., 2021 (link)), applying a RefAligner (Bionano Genomics) function simpleRepeatStandalone and repeat stretch tolerance of 0.1. Arrays of six and more repeat units were considered. Unit size estimates obtained from the optical map data were corrected using the coefficient of 0.952 to eliminate error due to 4.8% expansion of the optical map compared to the sequence, calculated from a sequence‐to‐map alignment. The resulting rmap file was analysed in Microsoft Excel and unit size and number of units were plotted in a histogram for visual analysis.
7
Optical Genome Mapping of Proband
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Optical genome mapping was performed on a genomic DNA sample from the proband using dual enzymes (BspQI, BssSI) on the Saphyr platform (BioNano Genomics, San Diego, CA, USA). Data was processed as described previously12 (link).
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