Fig. 3: Analysis of FGF14 repeat expansions in affected subjects using LR-PCR and nanopore sequencing.
From: Identification and characterisation of pathogenic and non-pathogenic FGF14 repeat expansions
A Gel electrophoresis of selected LR-PCR products spanning the FGF14 (AAG) STR locus. From left to right, M: 1 kb ladder; C: negative control; lanes 1-2: expansions between 220 and 299 repeats (1-M79607, 2-M90982); lane 3: individual M82415 shows one small and two large alleles; lanes 4-11: expansions above 300 repeats (4-M87668, 5-M84267, 6-M93354, 7-M81456, 8-M95289, 9-M80996, 10-M80920, 11-M96642 (937 repeats)). B Gel electrophoresis of LR-PCR products showing biallelic expansions: lanes 12-M96652, 13-M97638, 14-M95716, 15-M83825, 16-M80332, 17-M100781. M: 1 kb ladder; LR-PCR assays and gel electrophoresis were performed at least twice with the same results. C Waterfall plots showing selected nanopore reads after separation of alleles based on their flanking regions (methods). 300 randomly chosen reads are displayed in each graph. Blue: AAG repeats; Yellow: GAG; Red: AGG; Green: ACG; Pink: AAC; Black: other. Panel above: segregation of FGF14 alleles in family E19-1058 (three affected siblings, two with biallelic expansions and one with a single expansion of 325 repeats). Lower-left panel: individual M82415 (E20-0501) shows three different alleles: one small and two large (somatic mosaicism). Lower-right panel: individual M87859 (E21-0708) shows a pure AAG expansion (301 repeats) and a smaller allele with ACG interruptions. AAC streaks likely constitute errors of sequencing rather than true changes. D Nanopore reads from individual M81457 with an AAGGAG expansion. E Correlation between the median number of repeats detected by nanopore sequencing and expansion size estimated from fragment size analysis. F Standard deviation in allele size calculated from nanopore reads showing that somatic instability is positively correlated with expansion size. Blue: AAG; orange: AAGGAG main motif. For graphs (E and F), R2 is the square value of the Pearson correlation coefficient (two-sided) and 95% confidence intervals appear in light gray.
