The onset of the phylogenomic era has revolutionized molecular ecology and systematics, helping resolve relationships throughout the tree of life that have long eluded researchers working with only a handful of loci and morphological data. Phylogenetic studies of nonmodel organisms now routinely generate thousands to hundreds of thousands of loci to throw at a given question — despite the fact that only a fraction of these genes are necessary to fully resolve a tree in most cases. (And despite the fact that this glut of data can lead to major computational problems.)
However, the development of approaches intermediate between multiplex PCR and sequence capture / RAD-based methods has lagged behind the more extreme end of the spectrum. Where, then, does the biologist seeking to generate a reasonably-large-but-not-gratuitous number of loci turn? A new method known as Molecular Inversion Probes (MIPs) may provide an answer.
Figure 2 from Niedzicka et al. 2016: Molecular Inversion Probe structure and implementation.
As described in an article published in Nature Scientific Reports by M. Niedzicka and colleagues earlier this year, MIPs are 112 bp single-stranded nucleotides characterized by the presence of specific ligation and extension sequences that flank a target sequence of interest, and are bridged by “linker” sequence (Figure 1). During hybridization of MIPs to target DNA, gap-filling and ligation produce molecules containing the targeted sequence joined with adaptors and barcodes ready for downstream use.
Originally popular for use in biomedical research and human genome sequencing, Niedzicka et al. tested MIPs on the nonmodel salamanders Lissotriton montandoni and L. vulgaris. The team designed probes to target sequence across the genome from transcriptome data, focusing on regions that were diagnostic at the species level and identifying exon boundaries through a homology-based approach that relied on the conservation of these regions across vertebrates. Of 248 designed markers, 234 amplified successfully, and 80% of those had median coverage within one order of magnitude. Additionally, 77% of the MIPs were confirmed as single copy Mendelian markers, and replicate samples were genotyped identically with MIPs 99% of the time.
While the requirements of pre-existing transcriptome data and probe design keep MIPs from having the immediate “going in blind” ease of RADseq or UCEs with a published probeset, they nonetheless offer several concrete advantages over these methods. Unlike RADseq, for instance, MIPs require limited quantities of DNA, and have no ascertainment bias associated with evolutionary divergence between sequences. And unlike targeted capture, the lab protocol is relatively straightforward and cheap, with no need for the preparation of genomic libraries.
As the authors themselves put it: “We would thus like to bring the MIP markers to the attention of researchers as a useful extension of the molecular toolkit and an effective solution for large-scale resequencing of tens or hundreds of kb in ecological and evolutionary studies.” It will be interesting to see the first batch of empirical studies to use MIPs, and how widely they catch on among molecular ecologists.
References:
Ai, B., Kang, M. 2015. How Many Genes are Needed to Resolve Phylogenetic Incongruence? Evolutionary Bioinformatics Online. DOI: 10.4137/EBO.S26047
McCormack, J.E., Faircloth, B.C., Crawford, N.G., Gowaty, P.A., Brumfield, R.T., Glenn, T.C. 2012. Ultraconserved Elements Are Novel Phylogenomic Markers that Resolve Placental Mammal Phylogeny when Combined with Species Tree Analysis. Genome Research. DOI: 10.1101/gr.125864.111.
Niedzicka, M., Fijarczyk, A., Dudek, K., Stuglik, M., Babik, W. 2016. Molecular Inversion Probes for targeted resequencing in non-model organisms. Nature Scientific Reports. DOI: 10.1038/srep24051