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Development of T andem R epeat-based Identification of X -chromosome Inactivation (TRiXi) for high-sensitivity detection of skewed X-inactivation. a, forward (blue) and reverse (green) primer binding sites, repeat location (hg38), repeat <t>sequence,</t> PCR product GC content (%) and methylation of CpG sites in TRiXi 1-5 PCR target regions. Hpa II (CCGG) sites are highlighted in red. b , allele length distribution of all five TRiXi repeats estimated from 50 randomly selected female WGS samples from the GTEx dataset. The highly polymorphic AR (X-Linked) and HTT (autosomal) tandem repeats are shown for comparison. c , schematic illustration of the TRiXi method involving methylation-sensitive digest of the alleles on the active X-chromosome followed by multiplex PCR of all 10 alleles (five polymorphic loci) followed by fragment size determination by capillary electrophoresis. d , Representative results of a single TRiXi assay. Top and bottom panels show undigested and digested runs of a highly skewed female sample from the ABIS cohort (sample ABIS 1). e , Scatterplot of XCI skew of 50 neonatal samples determined by HUMARA and TRiXi. Spearman’s rank correlation test; ρ = 0.75, p = 7.1×10 −8 . f , Boxplot of XCI skew of 50 neonatal samples determined by HUMARA and TRiXi. Samples which were uninformative in HUMARA assay are indicated in grey. Two-tailed Wilcoxon rank-sum test; p = 0.68. e , f , light-blue dots highlight sample ABIS 1. g , Clustering of ONT reads by repeat length at all five repeat loci in the ABIS 1 sample. Color gradient indicates CpG <t>DNA</t> methylation level. Red arrows indicate CCGG sites.
Whole Genome Dna Sequencing Libraries, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Development of T andem R epeat-based Identification of X -chromosome Inactivation (TRiXi) for high-sensitivity detection of skewed X-inactivation. a, forward (blue) and reverse (green) primer binding sites, repeat location (hg38), repeat <t>sequence,</t> PCR product GC content (%) and methylation of CpG sites in TRiXi 1-5 PCR target regions. Hpa II (CCGG) sites are highlighted in red. b , allele length distribution of all five TRiXi repeats estimated from 50 randomly selected female WGS samples from the GTEx dataset. The highly polymorphic AR (X-Linked) and HTT (autosomal) tandem repeats are shown for comparison. c , schematic illustration of the TRiXi method involving methylation-sensitive digest of the alleles on the active X-chromosome followed by multiplex PCR of all 10 alleles (five polymorphic loci) followed by fragment size determination by capillary electrophoresis. d , Representative results of a single TRiXi assay. Top and bottom panels show undigested and digested runs of a highly skewed female sample from the ABIS cohort (sample ABIS 1). e , Scatterplot of XCI skew of 50 neonatal samples determined by HUMARA and TRiXi. Spearman’s rank correlation test; ρ = 0.75, p = 7.1×10 −8 . f , Boxplot of XCI skew of 50 neonatal samples determined by HUMARA and TRiXi. Samples which were uninformative in HUMARA assay are indicated in grey. Two-tailed Wilcoxon rank-sum test; p = 0.68. e , f , light-blue dots highlight sample ABIS 1. g , Clustering of ONT reads by repeat length at all five repeat loci in the ABIS 1 sample. Color gradient indicates CpG <t>DNA</t> methylation level. Red arrows indicate CCGG sites.
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Development of T andem R epeat-based Identification of X -chromosome Inactivation (TRiXi) for high-sensitivity detection of skewed X-inactivation. a, forward (blue) and reverse (green) primer binding sites, repeat location (hg38), repeat <t>sequence,</t> PCR product GC content (%) and methylation of CpG sites in TRiXi 1-5 PCR target regions. Hpa II (CCGG) sites are highlighted in red. b , allele length distribution of all five TRiXi repeats estimated from 50 randomly selected female WGS samples from the GTEx dataset. The highly polymorphic AR (X-Linked) and HTT (autosomal) tandem repeats are shown for comparison. c , schematic illustration of the TRiXi method involving methylation-sensitive digest of the alleles on the active X-chromosome followed by multiplex PCR of all 10 alleles (five polymorphic loci) followed by fragment size determination by capillary electrophoresis. d , Representative results of a single TRiXi assay. Top and bottom panels show undigested and digested runs of a highly skewed female sample from the ABIS cohort (sample ABIS 1). e , Scatterplot of XCI skew of 50 neonatal samples determined by HUMARA and TRiXi. Spearman’s rank correlation test; ρ = 0.75, p = 7.1×10 −8 . f , Boxplot of XCI skew of 50 neonatal samples determined by HUMARA and TRiXi. Samples which were uninformative in HUMARA assay are indicated in grey. Two-tailed Wilcoxon rank-sum test; p = 0.68. e , f , light-blue dots highlight sample ABIS 1. g , Clustering of ONT reads by repeat length at all five repeat loci in the ABIS 1 sample. Color gradient indicates CpG <t>DNA</t> methylation level. Red arrows indicate CCGG sites.
Genomic Dna, supplied by Zymo Research, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Development of T andem R epeat-based Identification of X -chromosome Inactivation (TRiXi) for high-sensitivity detection of skewed X-inactivation. a, forward (blue) and reverse (green) primer binding sites, repeat location (hg38), repeat <t>sequence,</t> PCR product GC content (%) and methylation of CpG sites in TRiXi 1-5 PCR target regions. Hpa II (CCGG) sites are highlighted in red. b , allele length distribution of all five TRiXi repeats estimated from 50 randomly selected female WGS samples from the GTEx dataset. The highly polymorphic AR (X-Linked) and HTT (autosomal) tandem repeats are shown for comparison. c , schematic illustration of the TRiXi method involving methylation-sensitive digest of the alleles on the active X-chromosome followed by multiplex PCR of all 10 alleles (five polymorphic loci) followed by fragment size determination by capillary electrophoresis. d , Representative results of a single TRiXi assay. Top and bottom panels show undigested and digested runs of a highly skewed female sample from the ABIS cohort (sample ABIS 1). e , Scatterplot of XCI skew of 50 neonatal samples determined by HUMARA and TRiXi. Spearman’s rank correlation test; ρ = 0.75, p = 7.1×10 −8 . f , Boxplot of XCI skew of 50 neonatal samples determined by HUMARA and TRiXi. Samples which were uninformative in HUMARA assay are indicated in grey. Two-tailed Wilcoxon rank-sum test; p = 0.68. e , f , light-blue dots highlight sample ABIS 1. g , Clustering of ONT reads by repeat length at all five repeat loci in the ABIS 1 sample. Color gradient indicates CpG <t>DNA</t> methylation level. Red arrows indicate CCGG sites.
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Development of T andem R epeat-based Identification of X -chromosome Inactivation (TRiXi) for high-sensitivity detection of skewed X-inactivation. a, forward (blue) and reverse (green) primer binding sites, repeat location (hg38), repeat <t>sequence,</t> PCR product GC content (%) and methylation of CpG sites in TRiXi 1-5 PCR target regions. Hpa II (CCGG) sites are highlighted in red. b , allele length distribution of all five TRiXi repeats estimated from 50 randomly selected female WGS samples from the GTEx dataset. The highly polymorphic AR (X-Linked) and HTT (autosomal) tandem repeats are shown for comparison. c , schematic illustration of the TRiXi method involving methylation-sensitive digest of the alleles on the active X-chromosome followed by multiplex PCR of all 10 alleles (five polymorphic loci) followed by fragment size determination by capillary electrophoresis. d , Representative results of a single TRiXi assay. Top and bottom panels show undigested and digested runs of a highly skewed female sample from the ABIS cohort (sample ABIS 1). e , Scatterplot of XCI skew of 50 neonatal samples determined by HUMARA and TRiXi. Spearman’s rank correlation test; ρ = 0.75, p = 7.1×10 −8 . f , Boxplot of XCI skew of 50 neonatal samples determined by HUMARA and TRiXi. Samples which were uninformative in HUMARA assay are indicated in grey. Two-tailed Wilcoxon rank-sum test; p = 0.68. e , f , light-blue dots highlight sample ABIS 1. g , Clustering of ONT reads by repeat length at all five repeat loci in the ABIS 1 sample. Color gradient indicates CpG <t>DNA</t> methylation level. Red arrows indicate CCGG sites.
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Development of T andem R epeat-based Identification of X -chromosome Inactivation (TRiXi) for high-sensitivity detection of skewed X-inactivation. a, forward (blue) and reverse (green) primer binding sites, repeat location (hg38), repeat <t>sequence,</t> PCR product GC content (%) and methylation of CpG sites in TRiXi 1-5 PCR target regions. Hpa II (CCGG) sites are highlighted in red. b , allele length distribution of all five TRiXi repeats estimated from 50 randomly selected female WGS samples from the GTEx dataset. The highly polymorphic AR (X-Linked) and HTT (autosomal) tandem repeats are shown for comparison. c , schematic illustration of the TRiXi method involving methylation-sensitive digest of the alleles on the active X-chromosome followed by multiplex PCR of all 10 alleles (five polymorphic loci) followed by fragment size determination by capillary electrophoresis. d , Representative results of a single TRiXi assay. Top and bottom panels show undigested and digested runs of a highly skewed female sample from the ABIS cohort (sample ABIS 1). e , Scatterplot of XCI skew of 50 neonatal samples determined by HUMARA and TRiXi. Spearman’s rank correlation test; ρ = 0.75, p = 7.1×10 −8 . f , Boxplot of XCI skew of 50 neonatal samples determined by HUMARA and TRiXi. Samples which were uninformative in HUMARA assay are indicated in grey. Two-tailed Wilcoxon rank-sum test; p = 0.68. e , f , light-blue dots highlight sample ABIS 1. g , Clustering of ONT reads by repeat length at all five repeat loci in the ABIS 1 sample. Color gradient indicates CpG <t>DNA</t> methylation level. Red arrows indicate CCGG sites.
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Development of T andem R epeat-based Identification of X -chromosome Inactivation (TRiXi) for high-sensitivity detection of skewed X-inactivation. a, forward (blue) and reverse (green) primer binding sites, repeat location (hg38), repeat <t>sequence,</t> PCR product GC content (%) and methylation of CpG sites in TRiXi 1-5 PCR target regions. Hpa II (CCGG) sites are highlighted in red. b , allele length distribution of all five TRiXi repeats estimated from 50 randomly selected female WGS samples from the GTEx dataset. The highly polymorphic AR (X-Linked) and HTT (autosomal) tandem repeats are shown for comparison. c , schematic illustration of the TRiXi method involving methylation-sensitive digest of the alleles on the active X-chromosome followed by multiplex PCR of all 10 alleles (five polymorphic loci) followed by fragment size determination by capillary electrophoresis. d , Representative results of a single TRiXi assay. Top and bottom panels show undigested and digested runs of a highly skewed female sample from the ABIS cohort (sample ABIS 1). e , Scatterplot of XCI skew of 50 neonatal samples determined by HUMARA and TRiXi. Spearman’s rank correlation test; ρ = 0.75, p = 7.1×10 −8 . f , Boxplot of XCI skew of 50 neonatal samples determined by HUMARA and TRiXi. Samples which were uninformative in HUMARA assay are indicated in grey. Two-tailed Wilcoxon rank-sum test; p = 0.68. e , f , light-blue dots highlight sample ABIS 1. g , Clustering of ONT reads by repeat length at all five repeat loci in the ABIS 1 sample. Color gradient indicates CpG <t>DNA</t> methylation level. Red arrows indicate CCGG sites.
Whole Genome Sequencing Genomic Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Development of T andem R epeat-based Identification of X -chromosome Inactivation (TRiXi) for high-sensitivity detection of skewed X-inactivation. a, forward (blue) and reverse (green) primer binding sites, repeat location (hg38), repeat <t>sequence,</t> PCR product GC content (%) and methylation of CpG sites in TRiXi 1-5 PCR target regions. Hpa II (CCGG) sites are highlighted in red. b , allele length distribution of all five TRiXi repeats estimated from 50 randomly selected female WGS samples from the GTEx dataset. The highly polymorphic AR (X-Linked) and HTT (autosomal) tandem repeats are shown for comparison. c , schematic illustration of the TRiXi method involving methylation-sensitive digest of the alleles on the active X-chromosome followed by multiplex PCR of all 10 alleles (five polymorphic loci) followed by fragment size determination by capillary electrophoresis. d , Representative results of a single TRiXi assay. Top and bottom panels show undigested and digested runs of a highly skewed female sample from the ABIS cohort (sample ABIS 1). e , Scatterplot of XCI skew of 50 neonatal samples determined by HUMARA and TRiXi. Spearman’s rank correlation test; ρ = 0.75, p = 7.1×10 −8 . f , Boxplot of XCI skew of 50 neonatal samples determined by HUMARA and TRiXi. Samples which were uninformative in HUMARA assay are indicated in grey. Two-tailed Wilcoxon rank-sum test; p = 0.68. e , f , light-blue dots highlight sample ABIS 1. g , Clustering of ONT reads by repeat length at all five repeat loci in the ABIS 1 sample. Color gradient indicates CpG <t>DNA</t> methylation level. Red arrows indicate CCGG sites.
Metagenomic Sequencing Genomic Dna Gdna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Development of T andem R epeat-based Identification of X -chromosome Inactivation (TRiXi) for high-sensitivity detection of skewed X-inactivation. a, forward (blue) and reverse (green) primer binding sites, repeat location (hg38), repeat sequence, PCR product GC content (%) and methylation of CpG sites in TRiXi 1-5 PCR target regions. Hpa II (CCGG) sites are highlighted in red. b , allele length distribution of all five TRiXi repeats estimated from 50 randomly selected female WGS samples from the GTEx dataset. The highly polymorphic AR (X-Linked) and HTT (autosomal) tandem repeats are shown for comparison. c , schematic illustration of the TRiXi method involving methylation-sensitive digest of the alleles on the active X-chromosome followed by multiplex PCR of all 10 alleles (five polymorphic loci) followed by fragment size determination by capillary electrophoresis. d , Representative results of a single TRiXi assay. Top and bottom panels show undigested and digested runs of a highly skewed female sample from the ABIS cohort (sample ABIS 1). e , Scatterplot of XCI skew of 50 neonatal samples determined by HUMARA and TRiXi. Spearman’s rank correlation test; ρ = 0.75, p = 7.1×10 −8 . f , Boxplot of XCI skew of 50 neonatal samples determined by HUMARA and TRiXi. Samples which were uninformative in HUMARA assay are indicated in grey. Two-tailed Wilcoxon rank-sum test; p = 0.68. e , f , light-blue dots highlight sample ABIS 1. g , Clustering of ONT reads by repeat length at all five repeat loci in the ABIS 1 sample. Color gradient indicates CpG DNA methylation level. Red arrows indicate CCGG sites.

Journal: medRxiv

Article Title: TRiXi: A Multi-Target Tandem Repeat-Based Method for Accurate Detection of X-Inactivation Skewing in Humans

doi: 10.1101/2025.11.24.25340860

Figure Lengend Snippet: Development of T andem R epeat-based Identification of X -chromosome Inactivation (TRiXi) for high-sensitivity detection of skewed X-inactivation. a, forward (blue) and reverse (green) primer binding sites, repeat location (hg38), repeat sequence, PCR product GC content (%) and methylation of CpG sites in TRiXi 1-5 PCR target regions. Hpa II (CCGG) sites are highlighted in red. b , allele length distribution of all five TRiXi repeats estimated from 50 randomly selected female WGS samples from the GTEx dataset. The highly polymorphic AR (X-Linked) and HTT (autosomal) tandem repeats are shown for comparison. c , schematic illustration of the TRiXi method involving methylation-sensitive digest of the alleles on the active X-chromosome followed by multiplex PCR of all 10 alleles (five polymorphic loci) followed by fragment size determination by capillary electrophoresis. d , Representative results of a single TRiXi assay. Top and bottom panels show undigested and digested runs of a highly skewed female sample from the ABIS cohort (sample ABIS 1). e , Scatterplot of XCI skew of 50 neonatal samples determined by HUMARA and TRiXi. Spearman’s rank correlation test; ρ = 0.75, p = 7.1×10 −8 . f , Boxplot of XCI skew of 50 neonatal samples determined by HUMARA and TRiXi. Samples which were uninformative in HUMARA assay are indicated in grey. Two-tailed Wilcoxon rank-sum test; p = 0.68. e , f , light-blue dots highlight sample ABIS 1. g , Clustering of ONT reads by repeat length at all five repeat loci in the ABIS 1 sample. Color gradient indicates CpG DNA methylation level. Red arrows indicate CCGG sites.

Article Snippet: Whole genome DNA sequencing libraries were prepared with the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, E7645S) and the NEBNext Multiplex Oligos for Illumina Index Primers Set 1 (New England Biolabs, E7335S).

Techniques: Binding Assay, Sequencing, Methylation, Comparison, Multiplex Assay, Electrophoresis, Two Tailed Test, DNA Methylation Assay