Figure 2
GATA1 mutation analysis in DS neonates with TAM and silent TAM. (A) Flow diagram of preparation and analysis of samples for deep sequencing. (B) Pie charts of GATA1 mutation analysis of the 200 babies in the cohort by standard Ss/DHPLC (left) and NGS (right). (C) Examples of base-pair plots from NGR analysis of patient samples (mutation indicated by arrows) with (D) corresponding pyrosequencing traces below (mutant peaks indicated by arrows). On the x-axis is the position along the GATA1 exon 2 amplicon (432 base pairs). On the y-axis is the read depth at different positions along the amplicon. Therefore, the black line trace shows the number of reads mapping to GATA1 sequence at different positions along the amplicon. At the position of the black arrowhead, a mutation was introduced into the PCR primer (mapping outside GATA1 exon 2) so that all PCR products would have a unique tag. This introduced mutation is detected by the blue line. All PCR products have this introduced mutation as the height of the blue line is to the level of black trace (total number of mapping reads). Sequence analysis also shows there are 2 common single-nucleotide polymorphism at positions rs62600348 T>G and rs66717003 T>G (indicated by the star) in the amplicon that map to position 48649449 and 48649456 within GATA1 exon 2. Nucleotide 0 is the first nucleotide of GATA1 exon 1 including exons and introns. NCBI reference NT_079573.4 (Homo sapiens chromosome X genomic contig, starting position 11496706) was used. The location of GATA1 mutation is indicated by the black arrow. (Ci,Di) Patient sample DST11 with a 7 bp duplication at position 48649625 previously detected by Ss/DHPLC. (Cii,Dii) Patient sample DST9 with an insertion of 7 bp at position 48649670 previously detected by DHPLC only. (Ciii,Diii) Patient sample DS158 with a 2 bp deletion at position 48649552 detected by NGS only and confirmed by pyrosequencing. (Civ,Div) Patient sample DS79 with a point mutation at position 48649565 detected by NGS only but not detectable by pyrosequencing. (E) Relationship between GATA1 mutant clone size (y-axis) as determined by NGR and % blasts detected by morphology. (F) Distribution of % blasts in TAM (n = 17) (filled red circles, left), silent TAM (n = 88) (open red cell circles, middle) and in samples without a GATA1 mutation detected by NGR (n = 70) (filled black circles, right).

GATA1 mutation analysis in DS neonates with TAM and silent TAM. (A) Flow diagram of preparation and analysis of samples for deep sequencing. (B) Pie charts of GATA1 mutation analysis of the 200 babies in the cohort by standard Ss/DHPLC (left) and NGS (right). (C) Examples of base-pair plots from NGR analysis of patient samples (mutation indicated by arrows) with (D) corresponding pyrosequencing traces below (mutant peaks indicated by arrows). On the x-axis is the position along the GATA1 exon 2 amplicon (432 base pairs). On the y-axis is the read depth at different positions along the amplicon. Therefore, the black line trace shows the number of reads mapping to GATA1 sequence at different positions along the amplicon. At the position of the black arrowhead, a mutation was introduced into the PCR primer (mapping outside GATA1 exon 2) so that all PCR products would have a unique tag. This introduced mutation is detected by the blue line. All PCR products have this introduced mutation as the height of the blue line is to the level of black trace (total number of mapping reads). Sequence analysis also shows there are 2 common single-nucleotide polymorphism at positions rs62600348 T>G and rs66717003 T>G (indicated by the star) in the amplicon that map to position 48649449 and 48649456 within GATA1 exon 2. Nucleotide 0 is the first nucleotide of GATA1 exon 1 including exons and introns. NCBI reference NT_079573.4 (Homo sapiens chromosome X genomic contig, starting position 11496706) was used. The location of GATA1 mutation is indicated by the black arrow. (Ci,Di) Patient sample DST11 with a 7 bp duplication at position 48649625 previously detected by Ss/DHPLC. (Cii,Dii) Patient sample DST9 with an insertion of 7 bp at position 48649670 previously detected by DHPLC only. (Ciii,Diii) Patient sample DS158 with a 2 bp deletion at position 48649552 detected by NGS only and confirmed by pyrosequencing. (Civ,Div) Patient sample DS79 with a point mutation at position 48649565 detected by NGS only but not detectable by pyrosequencing. (E) Relationship between GATA1 mutant clone size (y-axis) as determined by NGR and % blasts detected by morphology. (F) Distribution of % blasts in TAM (n = 17) (filled red circles, left), silent TAM (n = 88) (open red cell circles, middle) and in samples without a GATA1 mutation detected by NGR (n = 70) (filled black circles, right).

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