Bionano Technology at the FGCZ
Table of contents
If you are interested in using this technology at the FGCZ, please contact:
- Dr. Lucy Poveda (email@example.com) , Andrea Patrignani (firstname.lastname@example.org)
- Dr. Giancarlo Russo (email@example.com)
- 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
- 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 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
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.
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.
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.
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.