Targeted Capture and High-Throughput Sequencing Using Molecular Inversion Probes (MIPs)
Molecular inversion probes (MIPs) in combination with massively parallel DNA sequencing represent a versatile, yet economical tool for targeted sequencing of genomic DNA. Several thousand genomic targets can be selectively captured using long oligonucleotides containing unique targeting arms and universal linkers. The ability to append sequencing adaptors and sample-specific barcodes allows large-scale pooling and subsequent high-throughput sequencing at relatively low cost per sample. Here, we describe a “wet bench” protocol detailing the capture and subsequent sequencing of >2000 genomic targets from 192 samples, representative of a single lane on the Illumina HiSeq 2000 platform.
Key wordsMolecular inversion probes Massively parallel sequencing Real-time PCR Exonuclease cleanup and gel electrophoresis
We thank Bradley P. Coe for his critical review of the manuscript and Tonia Brown for assistance with the manuscript preparation. We thank Brian J. O’Roak, Beth Martin, Evan A. Boyle, and Joseph B. Hiatt for their overall contributions to developing the MIP protocol. S.C. is supported by a National Health and Medical Research Council (NHMRC) CJ Martin Biomedical Fellowship (#1073726). H.A.S. is supported, in part, by the NHGRI Interdisciplinary Training in Genome Science Grant (T32HG00035). E.E.E. is an investigator of the Howard Hughes Medical Institute. J.S. is an investigator of the Howard Hughes Medical Institute.
Competing financial Interests
E.E.E. is on the scientific advisory board (SAB) of DNAnexus, Inc., and is a consultant for the Kunming University of Science and Technology (KUST) as part of the 1000 China Talent Program.
- 8.O'Roak BJ, Stessman HA, Boyle EA et al (2014) Recurrent de novo mutations implicate novel genes underlying simplex autism risk. Nat Commun 5(5595):1–6Google Scholar
- 9.Qubit® Assays: Quick Reference Guide: Pub.no: MAN0010876Google Scholar
- 10.Qubit® 2.0 Fluorometer: MAN0003231Google Scholar
- 11.MiSeq: Denature and Dilute Libraries Guide: 15039740v1Google Scholar
- 12.MiSeq: Reagent Kit v3- Preparation Guide: Part#15044983Google Scholar
- 13.MiSeq System User Guide: part # 15027617Google Scholar