Benutzerspezifische Werkzeuge

Molecular Inversion Probes (smMIPs)

single molecule Molecular Inversion Probes (smMIPs)                

smMIPs  is a very cost-effective capture method of the genomic regions of interest and reprensets a modification of the molecular inversion probes (MIP) technology resulting in a highly sensitive variant calling and precise quantitation of mutation frequency, therefore addressing not only germline but also postzygotic somatic mutations [3]. MIPs are single stranded DNA molecules and contain sequences that are complementary to the target in the genome, and therefore can hybridize and capture the genomic target. MIP probes share the common design of two genomic target complementary segments, separated by a linker region. When the probe hybridizes to the target, it undergoes an inversion in configuration, circularizes, and the internal linker region forms a free hanging loop.The principle of smMIP is depicted in the following Figure of Hiatt, JB et al.: Single molecule molecular inversion probes for targeted, high-accuracy detection of low-frequency variation. Genom Res (2013), doi: 10.1101/gr.147686.112..

Figure. Principle of smMIP: (A) The MIP consists of two “targeting arms” (complementary to the target in the genome), joined by a constant “backbone” sequence containing a stretch of 12 randomly chosen nucleotides or “molecular tags” that can generate 1.67x106 unique sequences. (B) After hybridization to genomic DNA, the adding of polymerase and ligase leads to “gap-filling” of the reverse complement of the genomic DNA (copied target sequence); the probe is ligated into a single-stranded circle. (C) After exonuclease treatment and PCR, sequencing library molecules consist of platform compatibility (black), probe backbone (light gray), targeting arm (dark gray), copied target (light blue), molecular tag (red), and sample-specific index introduced during PCR (green). Massively parallel sequencing is used to collect three reads (dark blue). (D) Overlapping read-pairs are reconciled to form ‘‘fr-reads’’ (dark blue), assigned to samples via the sample-specific index sequence (green) and individual capture events via the molecular tag (red). (E) Groups of fr-reads assigned to the same probe via alignment to the reference genome and sharing the same molecular tag and sample index form a ‘‘tag-defined read group’’ (TDRG). Random errors (yellow) that occur during library construction and sequencing may be present in some members of the TDRG at some positions. (F) TDRGs are used to call a single molecule consensus sequence (‘‘smc-read’’) that is robust to such errors.