<b>Rapid</b><b>Analysis of </b><b>FMR1</b><b>5'UTR CGG Repeat Expansions</b><b>by long-read PCR and </b><b>Oxford Nanopore Third Generation Sequencing</b>

<b>Background:</b> Conventional genetic testsfor fragile X syndrome still presentedsome limitations, such ascomplicated proceduresand large sample amount for Southern blot, and limitied mutation types for triplet repeat-primed polymerase chain reaction(TP-PCR). Currently,third-generation long-read sequencing is a potential technology for thesimultaneous detection of CGG repeat expansions, point mutations, and deletions. However, no simple method exists for obtainingthe target long-fragment CGG repeat region with ultra-high GC content forthird-generationsequencing.<b>Methods:</b><b> </b>We developed a rapid long-fragment ultra-high GC polymerase chain reaction (PCR) amplificationapproach to directly prepare long-readlibraries, followed by Oxford Nanopore sequencingto detect the full spectrum of FMR1 5′UTR CGG repeat mutations. This approach was applied to 10 standard cell line samples, 53 retrospectiveclinical blood samples, and 9 prenatal amniotic fluid samples.<b>Results:</b> We found that the FMR15’UTR CGG expansion genotypes identified usingour approach were completely consistentwith the results of TP-PCR inall10 cell line samples,53 clinical samples, and 9 prenatal amniotic fluid samples. The recognizable number of CGG repeatsin normal and pre-mutation types was also significantly correlated(male cell lines, n=3×triplicate, R2=0.9996; femalecell lines, n=4×triplicate, R2=0.9972; clinical male blood samples, n=11, R2=1.0000; clinical femalebloodsamples, n=22, R2=0.9854).<b>Conclusion: </b>This studyprovided a simple andcost-effective approachbased on long-fragment ultra-high GC PCRto obtain the FMR1 5′UTR CGG repeatregion and direct library for Oxford Nanopore Third Generation Sequencing, and it may be used in Fragile X syndrome diagnosis and prenatal care, and as a reference method for detecting other previously unsolved diseases caused by short tandem repeat expansions with ultra-high GC content, such as myotonic dystrophy and Huntington’ s disease.