Bionano Technology at the FGCZ
Table of contents
Access at the FGCZThis platform is available at the FGCZ through a user-lab access, ,meaning that we will provide support and the reagents for you to conduct your experiments userlab-access.
If you are interested in using this technology at the FGCZ, please contact:
- Dr. Lucy Poveda (firstname.lastname@example.org) , Andrea Patrignani (email@example.com)
- Dr. Giancarlo Russo (firstname.lastname@example.org)
Main applicationsWe use the Bionano Saphyr Chip version G1.2 on the Bionano Saphyr platform.This version of the Saphyr chip has 2 independent compartments or flowcells, each of which can deliver 1,300 Gbp of raw data (molecules with a length ≥ 150,000bp). Depending on the size of the genome and the coverage required, one can run 2 samples simultaneously on 1 Chip (1 sample/ flowcell) or 1 sample in 1 Chip (1 sample/chip).
I) Hybrid Scaffolding for Genome Assembly
- Enhance & refine genome references and draft genomes
- Orient and align sequencing reads using a hybrid scaffold
- Close the contig gaps (Example 2500 contigs down to 300-100)
- Combine with sequencing reads for de novo assembly in species without a reference genome
- Comparative Genomic analysis
II) Long range Structural Variant Discovery and Detection
- Coverage required for SV detection: 80-100 or 300-400x coverage is recommended, depending on whether one runs the variant analysis pipeline or the rare variant pipeline, respectively
Bionano Whole Genome Imaging Technology and Experimental Information
Bionano Whole Genome Imaging is an optical mapping platform that enables both structural variant identification as well as scaffolding of genome de novo assemblies. This platform provides a streamlined workflow starting from the sample/tissue to the analysis of the data.
The workflow of a Bionano experiment can be spilt into the following steps:
a) High molecular weight DNA extraction
b) Labeling of HMW DNA
d) Data analysis
a) High molecular weight DNA extractionThe aim is to isolate ultra-high molecular weight DNA from fresh or frozen blood, cultured cells, and plant and animal tissues by using specific kits. Unlike standard DNA isolation methods such as precipitation, column, or bead-based methods, which yield molecules that are rather small, Bionano’s sample prep method produces molecules, greater than 150,000 bp, which will then be used either to assemble or aligned against a reference.
Currently, there are two methods to isolate ultra-high molecular weight DNA: a solution-based method called Bionano Prep SP protocol that currently only works with blood and cells, and a method based on agarose gel plug DNA isolation which can work with most materials.
-This Bionano Prep SP Blood and Cell Culture DNA Isolation Kit can provide ultra-high molecular weight DNA in less than 4 hours for EDTA-collected blood and mammalian cell cultures. It utilizes a lyse, bind, wash, and elute procedure that is common for silica-based DNA extraction technologies in combination with a novel paramagnetic disk. Unlike magnetic beads and silica spin columns, which shear large DANN molecules, the Nanobind Disk binds and releases DNA with significantly less fragmentation.
-For ultra-high molecular weight DNA isolation from plant, animal and human tissues, a method based on agarose gel plug DNA isolation is available. These protocols are based on the isolation of cells or nuclei in an agarose matrix, where DNA purification takes place while the molecules are stabilized in agarose. By the end of the purification process, the agarose is digested and molecules up to chromosome arm lengths in size are released.
b) Labeling of HMW DNAOnce we have the HMW DNA isolated, then it is labeled at specific sequence motifs for imaging and identification. The labeling results in a uniquely identifiable sequence-specific pattern of labels in the DNA molecule that is then used for de novo map assembly as well as the alignment to the reference.
There are two labeling chemistries available, a Direct Labeling chemistry and a predecessor based on nicking endonucleases. The choice of the labeling chemistry and enzyme to be used will depend on the underlying genome sequence, for de novo assembly applications this is based on the NGS draft.
- Direct Label and Stain (DLS), recognises a 6 base-pair-sequence-motif and transfers a fluorescent label directly to it in a single enzymatic step.
- The predecessor, Nick-Label-Repair and Stain (NLRS) chemistry, is a nickase-based labeling. A nicking endonuclease creates a single-strand nick in the DNA molecules at a specific recognition sites, followed by incorporation of fluorescently labeled nucleotide analogs. NLRS chemistry leverages many commercially available enzymes to provide greater flexibility of target sequences.
c) ImagingA labeled DNA sample is transferred onto the Bionano Saphyr Chip™ in one of the compartments or so-called flowcells. The DNA will be electrophoretically moved and gently unwinded into the NanoChannels of the Bionano Saphyr Chip™. These NanoChannels allow only a single linearized DNA molecule to travel through while preventing the molecule from tangling or folding back on itself. The nanofluidic environment allows molecules to move swiftly through hundreds of thousands of parallel NanoChannels simultaneously, enabling high-throughput processing to build an accurate Bionano genome map.
Once the DNA is stretched inside the NanoChannels, the high-resolution Bionano Saphyr camera images them. Long molecules spanning beyond a field of view are stitched together. Once imaged, the molecules are flushed and the process is repeated, enabling imaging of more than 25 Gbp of DNA per hour per flowcell. In our Bionano Saphyr System we can run 1 Bionano Saphyr Chip™ at a time, each chip contains two compartments or flowcells, and thus allows to run two samples simultaneously. Per compartment/flowcell we can now aim to obtain 1300Gb of data. Typically for de novo assembly workflows, 70-100x effective coverage is recommended. For structural variant applications 80-100 or 300-400x coverage is recommended, depending on whether one runs the variant analysis pipeline or rare variant pipeline, respectively.
d) Data analysisOnce raw image data of labeled DNA molecules is captured by the Bionano Saphyr instrument, it is converted into digital representations of the motif-specific label pattern. Bionano Solve™ data analysis software then assembles the data de novo to recreate a consensus whole genome map assembly.
Bionano genome maps enable a variety of analyses, including hybrid scaffolding and structural variation detection. Bionano assemblies are not guided by a reference, allowing for unbiased reconstruction of the genome structure spanning.
Furthermore, for human at the moment only, it is also possible to align the molecules directly against the human reference to detect low frequency variants and by this avoiding building a consensus map.