BioNano genome mapping service at KSU
What are BioNano genomic maps?
BioNano genomic maps are whole genome restriction maps based on imaging ultra long >150kb single molecules of DNA that are fluorescently labeled at specific restriction sites using the methodology optimized by BioNano Genomics (BNG) San Diego CA USA. These maps can be compared to restriction maps inferred from a genome sequence assembly to validate the assembly, improve contiguity and identify structural variation.
There are several issues to take into account when considering this approach.
1) Do you have a genome sequence assembly? If not, it will be harder to determine which enzyme to nick the DNA with, there will be no way to QC the initial results, and there will be no reference to seed the assembly of BNG optical maps. We can try a denovo assembly in the absence of sequence data, but this will require deeper coverage (>100x) and will take considerably more compute hours.
2) If you do have an assembly, what condition is it in? This approach will not provide much benefit for highly fragmented genome assemblies. It is best if the contig N50 is >50kb and the scaffold N50 is >300kb. The DNA will be labeled at restriction sites that occur ~10 time per 100kb. If the sequence assembly scaffolds are short (<50kb), or contain only a few restriction sites, it is not possible to accurately align them with the BNG maps. We start with 1Mb DNA, and use nickases (specially adapted restriction enzymes that nick one strand of DNA (New England Biolabs)) to label the HMW DNA at specific sites (7mer restriction recognition sites). After incorporating florescent nucleotides, the nick is repaired and the DNA is electrophoresed into massively parallel nanochannels to be imaged.
3) Can you isolate ultra High Molecular Weight DNA from your organism? BNG has isolation protocols for supported samples including white blood cells or certain tissue culture lines from humans and other primates; red blood cells from birds, amphibians and reptiles; and bacteria. We have experience working with arthropods and plants. While these are not BNG supported samples, we will work with a client to optimize the recovery of HMW DNA.
4) Plants present additional challenges since they contain lots of carbohydrates, polyphenols, lecithin and other nasty things that complicate the recovery of pure, clean, intact HMW DNA. So one must start with very young tissue, usually immature leaves, that have been dark treated (usually 72 hours or until they start to turn yellow) to reduce the carbohydrates, unfortunately this usually induces a stress response, so it is important to experiment with this parameter. Please consider how many plants you will need to dark treat to harvest 5-10 grams of tissue. We can work with fresh or flash frozen tissue. Please note: We cannot overstate the importance of the condition of the initial sample material. There is no way to improve upon poor quality sample tissue.
Our fee-for service center provides Irys automated imaging (BioNano Genomics) to produce next generation maps of your favorite genome. To learn more about how this technology works, visit the BioNano Genomics website.
We can start with tissue samples (simple, homogenous tissue works best eg., amphibian red blood cells, mammalian white blood cells, cell lines) or high molecular weight DNA in plugs of low melt agarose. The following fee schedule details charges for each step in the process.
|Description of the Products or Services||Price|
|Experimental design and project setup||$570|
|Isolation of ultra long, ultra clean HMW DNA||each||$1073|
|Quantification and QC||each||$393|
|NickLabelRepair run (NLR)||each||$503|
|Compute maps and align with reference genome||hr||$114|
For a ~100Mb genome we would run a single flow cell (there are 2 flow cells per chip), larger genomes require more flow cells. Some clients isolate ultra pure HMW DNA in agarose (following detailed protocols we provide) and send the plugs to us (following detailed shipping instructions we provide). Larger genomes require additional NLRs and imaging.
For example, a quote for a 500Mb genome that required additional work to optimize the HMW DNA isolation might look like this:
price per unit ($)
|project set up||1||570||570|
Request an estimate:
Please provide the following information to receive a quote for genome maps of your favorite organism.
Send emails to firstname.lastname@example.org:
- Scientific name of organism (required)
- Name of project PI (required)
- Email address (required)
- Sequence data (required)
- Fasta file of your genome assembly and (if available) your scaffolds
- If sequence data is not available we can estimate nicking rate based on a close relative. NCBI has a genome database. Please send the link to a closely related genome with the most complete genome.
- If available, descriptive statistics of your genome and/or assembly (e.g. text files with number of contigs/scaffolds and cumulative length of assembly, or flow cytometry estimates of genome size).
Watch a BioNano Webinar "Using BioNano Maps to Improve an Insect Genome Assembly":