MOLECULAR INVERSION PROBES
Simple, Flexible Targeted NGS
Molecular Inversion Probes (MIP) allows adjustable targeting of specific regions of the genome with a simple workflow. The MIP design is a pair of single stranded DNA molecule probes that contain sequences complementary to the target, joined by a loop linker. The loop ensures that the probes bind in close proximity to each other, significantly reducing off-target probe binding. PCR is then used to close the circle by copying the target region between the 5’ and 3’ probes.
Key Advantages:
Workflow much simpler than hybridization enrichment– only 4 touch-points, sample prepared in a single tube/well so no need for sample transfer tracking
Fast to design – most new designs ship within 6 weeks
No upper limit on panel size
Consistent uniform and deep coverage, and high accuracy from overlapping probe design
Easy to use – color-coded reagents
UMIs and UDIs standard
Easy to automate and readily scalable
Suited for working with low quantities of DNA
*Figure 1. Summary of the Molecular Inversion Probe workflow
Figure 2. Comparison of workflows between molecular inversion probes and hybridization capture.
Custom NEXTFLEX® Panels
Custom panels can be designed to genes, breakpoints and CNVs. The design can be augmented with amplicons for pseudogenes, fuzzy breakpoints and short repeat expansions.
Example Custom NEXTFLEX® Extended Carrier Panel
Contains redundantly tiled Molecular Inversion Probes (MIPs) for 1722 targets. Targets include:
- Complete coding exonic sequences of 89 genes, at least 15 bases of exon-adjacent intronic sequences, and Clinvar database-specified pathogenic/likely pathogenic mutations located in introns or promoters of these genes,
- Mutation hotspots in additional 16 genes,
- Breakpoint probes for nine large deletions and one duplication,
- Selected mutations in 11 genes relevant to infertility conditions and microdeletions in three Azoospermia Factor regions of chromosome Y.
| NEXTFLEX® Extended Carrier Panel | |
|---|---|
| Target Size | 291,211 bp |
| Hands on Time | 1 Hour |
| Sample Prep Time | 3.5 Hours + Overnight hyb |
Figure 3. IGV view of NGS reads from overlapping molecular inversion probe design
Molecular Inversion Probe Performance on Genomic DNA (Average 0.9×106 Reads per Sample)
Figure 4a. Fraction of targets with depth of coverage ranging from 1x up to 100x
Consistently high depth of coverage across the target regions, with >99% of targeted bases having >100x depth
Figure 4b. Fold 80 base penalty across the targeted regions
Even coverage across the targeted regions, with a fold 80 base penalty averaging less than 1.76.
Figure 4c. Average depth of coverage across all 1,722 targets
Greater than 97.5% of targets generated greater than 30x coverage. Further customization of the probe pool is possible.
Molecular Inversion Probes Performance on Limited Template (2×106 reads)
Standard MIP kit performance on whole genome amplified cells.
Figure 5. Target specificity and depth of coverage from single cell and 5 cell whole genome amplified templates, compared to unamplified genomic DNA using a standard molecular inversion probe panel and protocol.
With no prior optimisation and using a standard genomic DNA kit and workflow, greater than 95% of the sequencing data was from bases on target or within 250bp up or downstream. Greater than 55% of targets from single cell templates and 70% of targets from 5 cell templates obtained at least 20x depth of coverage. This equates to 950 target regions from single cells and 1,224 target regions from 5 cells. A high sensitivity kit is also available.