Optical mapping
Optical mapping is a variation on restriction mapping, which can map the location of restriction enzyme sites along the unknown DNA of an organism. The spectrum of fragments from restriction enzyme digestion collectively serve as an unique 'fingerprint' or 'barcode' for that sequence. High-throughput platforms for producing optical maps have been produced by companies like Opgen. An improvement on optical mapping uses nanocoding technology [1] to trap elongated DNA molecules in nanoconfinements to improve single molecule genome analysis.
How Optical Mapping Works
Optical mapping utilizes the ability of restriction enzymes to recognize specific sequences in a genome of interest.
Uses
Optical mapping system has been used to construct whole-genome restriction maps of bacteria, parasites, fungi, maize. [2][3][4]
Optical Mapping Technologies
- DNA Sequencing
- Optical Sequencing
- Combining optical mapping with NGS platforms
- Comparative Genomics
Optical Sequencing
Optical sequencing is a single molecule DNA sequencing technique that follows sequence-by-synthesis and uses optical mapping technology[5][6]. Similar to other single molecular sequencing approaches such as SMRT sequencing, this technique analyzes a single DNA molecule, rather than amplify the initial sample and sequence multiple copies of the DNA. During synthesis, fluorochrome-labeled nucleotides are incorporated through the use of DNA polymerases and tracked by fluorescence microscopy. This technique was originally proposed by David C. Schwartz and Arvind Ramanathan in 2003.
The following is an overview of each cycle in the optical sequencing process. [7].
Step 1: DNA Barcoding
Cells are lysed to release genomic DNA. These DNA molecules are untangled, placed onto optical mapping surface containing microfluidic channels and the DNA is allowed to flow through the channels. These molecules are then barcoded by restriction enzymes to allow for genomic localization through the technique of optical mapping.
Step 2: Template Nicking
DNase I is added to randomly nick the mounted DNA molecules. A wash is then performed to remove the DNase I. The mean number of nicks that occur per template is dependent on the concentration of DNase I as well as the incubation time.
Step 3: Gap Formaton
T7 exonuclease is added which uses the nicks in the DNA molecules to expand the gaps in a 5'-3' direction. Amount of T7 exonuclease must be carefully controlled to avoid overly high levels of double-stranded breaks.
Step 4: Fluorochrome incorporation
DNA polymerase is used to incorporate fluorochrome-labelled nucleotides (FdNTPs) into the multiple gapped sites along each DNA molecule. During each cycle, the reaction mixture contains a single type of FdNTP and allows for multiple additions of that nucleotide type. Various washes are then performed to remove unincorporated fdNTPs in preparation for imaging and the next cycle of FdNTP addition.
Selection of an appropriate DNA polymerase is critical to the efficiency of the base addition step and must meet several criteria:
- Ability to efficiently incorporate FdNTP at consecutive positions
- Lack of 3'-5' exonuclease and proofreading activity to prevent the removal newly incorporated FdNTP
- High fidelity to minimize mis-incorporations
- Good activity on templates which are mounted to surfaces (eg. optical mapping surface)
In addition, different polymerase preference for different fluorochromes, linker length on fluorochrome-nucleotides, and buffer compositions are also important factors to be considered to optimize the base addition process and maximize number of consecutive FdNTP incorporations.
Step 5: Imaging
This step counts the number of incorporated fluorochrome-labeled nucleotides at the gap regions using fluorescence microscopy.
Step 6: Photobleaching
The laser illumination that is used to excite the fluorochrome is also used here to destroy the fluorochrome signal. This essentially resets the fluorochrome counter, and prepares the counter for the next cycle. This step is a unique aspect of optical sequencing as it does not actually remove the fluorochrome label of the nucleotide after its incorporation. not removing the fluorochrome label makes sequencing more economical, but it results in the need to incorporate fluorochrome labels consecutively which can result in problems due to the bulkiness of the labels.
Step 7: Repeat steps 4-6
Steps 4-6 are repeated with step 4 using a reaction mixture that contains a different fluorochrome-labeled nucleotide (FdNTP) each time. This is repeated until the desired region is sequenced.
Advantages
- minimal DNA sample required
- streamline sample preparation process
Large DNA molecule templates (~500 kb) vs. Short DNA molecule templates (< 1kb) While most next generation sequencing technologies aim of massive amounts of smalls sequence reads, these small sequence reads make de novo sequencing efforts and genome repeat regions difficult to comprehend. Optical sequencing uses large DNA molecule templates (~500 kb) for sequencing and these offers several advantages over small templates:
- These large DNA templates can be "DNA barcoded" to determine their genomic localization with confidence. Therefore, any sequence reads that are taken from the large template can be mapped onto the genome with a high degree of confidence. More importantly, sequence reads from high repeat regions can placed with a greater degree of confidence where as the short reads suffer from mapping uncertainty in high repeat regions. Special algorithms and software such as optical mapping and nanocoding have been developed to align single-molecule barcodes with a reference genome.
- Multiple sequence reads from the same large template molecule. These multiple sequence reads reduce the complexity of de novo assembly, disambiguate genomic rearrangement regions, and "intrinsically free from any assembly errors."[7]
- Molecular barcoding of large DNA molecular templates with sequence acquisition provides broad and specific genomic analyses
Concerns
- Single molecule DNA sequencing requires a high level of precision to match the confidence from the redundant read coverage provided by current next-generation sequencing technologies.
- Nicks on both strands at similar positions resulting in low template during sequence-by-synthesis.
- Fluorochrome-labeled nucleotides are not removed after incorporation and because of these bulky labels, multiple incorporation might be difficult.
Optical Mapping with Short-read NGS
Optical mapping has been used to
External Links
References
- ^ Kyubong Jo et al. (2007) "A single-molecule barcoding system using nanoslits for DNA analysis." PNAS, 104(8), 2673-2678.
- ^ Lai Z et al. "A shotgun optical map of the entire Plasmodium falciparum genome (1999). Nature Genetics, 23, 309-313.
- ^ Lim A et al. Shotgun optical maps of the whole Escherchia coli O157:H7 genome (2001). Genome Research, 11, 1584-1593.
- ^ Lin J et al. Whole-genome shotgun optical mapping of Deinococcus radiodurans (1999). Science, 285, 1558-1562.
- ^ Ramanathan, Arvind, et al. "An Integrative Approach for the Optical Sequencing of Single DNA Molecules." Analytical Biochemistry 330.2 (2004): 227-41.
- ^ Ramanathan, Arvind, Louise Paper, and David C. Schwartz. "High-Density Polymerase-Mediated Incorporation of Fluorochrome-Labeled Nucleotides." Analytical Biochemistry 337.1 (2005): 1-11.
- ^ a b Zhou, Shiguo, Louise Paper, and David C. Schwartz. "Optical Sequencing: Acquisition from Mapped Single-Molecule Templates." Next-Generation Genome Sequencing: Towards Personalized Medicine. Ed. Michal Janitz. 1st ed.Wiley-VCH, 2008. 133-151.