Homo sapiens Genome sequencing

In vitro fertilization (IVF), preimplantation genetic diagnosis (PGD), andpreimplantation genetic screening (PGS) help patients to select embryos free of monogenic diseases and aneuploidy (chromosomeabnormality). Next-generation sequencing (NGS) methods, while experiencing a rapid cost reduction, have improved the precision of PGD/PGS. However, the precision of PGD has been limited by the falsepositive and false-negative single-nucleotide variations (SNVs), whichare not acceptable in IVF and can be circumvented by linkage analyses,such as short tandem repeats or karyomapping. It is noteworthy thatexisting methods of detecting SNV/copy number variation (CNV) andlinkage analysis often require separate procedures for the sameembryo. Here we report an NGS-based PGD/PGS procedure that cansimultaneously detect a single-gene disorder and aneuploidy and iscapable of linkage analysis in a cost-effective way. This method, called“mutated allele revealed by sequencing with aneuploidy and linkageanalyses”(MARSALA), involves multiple annealing and looping-basedamplification cycles (MALBAC) for single-cell whole-genome amplification. Aneuploidy is determined by CNVs, whereas SNVs associated withthe monogenic diseases are detected by PCR amplification of theMALBAC product. The false-positive and -negative SNVs are avoidedby an NGS-based linkage analysis. Two healthy babies, free of themonogenic diseases of their parents, were born after such embryoselection. The monogenic diseases originated from a single basemutation on the autosome and the X-chromosome of the diseasecarrying father and mother, respectively.