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
Molecular Inversion 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:

  1. 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,
  2. Mutation hotspots in additional 16 genes,
  3. Breakpoint probes for nine large deletions and one duplication,
  4. 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
Molecular Inversion
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)

Molecular Inversion
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

Molecular Inversion
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.

Molecular Inversion
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.

Molecular Inversion
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.

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