Journal: American Journal of Human Genetics

Article Title: RNA variant assessment using transactivation and transdifferentiation

doi: 10.1016/j.ajhg.2024.06.018

Figure Lengend Snippet: Investigation of RNA variants in SMGs using transactivation of HDFs (A) Illustration of USH2A mRNA expression (red) in human adult tissues as reported by the Human Protein Atlas (HPA). (B) Diagram depicts the USH2A variant under investigation. Sashimi plots report USH2A mRNA splicing. Data derived from Oxford Nanopore long read sequencing of RT-PCR amplicons (exons 13–16) produced using RNA isolated following transactivation of USH2A in HDFs derived from healthy control and affected individuals in the presence and absence of cycloheximide (CHX). Arrows in the sashimi plots specify the reads coming from the alleles with pathogenic variant and allele with VUS as segregated by allelic phasing. (C) Illustration of SCN1A mRNA expression (red) in human adult tissues (HPA). (D) Diagram depicts the SCN1A variant under investigation. Sashimi plots report SCN1A mRNA splicing. Data derived from Oxford Nanopore long-read sequencing RT-PCR amplicons (exons 13–17) produced from RNA isolated following transactivation of SCN1A in HDFs derived from healthy control and affected individuals in the presence and absence of CHX. Arrows on the sashimi plot indicates the position of the pathogenic variant. (E) Illustration of DMD mRNA expression (red) in human adult tissues (HPA). (F) Diagram depicts the DMD variant under investigation. Graphs represent relative read depth of reported across DMD exons 2–5 as determined using long read sequencing of RT-PCR amplicons produced from RNA isolated following transactivation of DMD in HDFs derived from healthy control and affected individuals in the presence and absence of CHX. Note read depth is 1.8 times greater (∼double) in exons 3– and 4 only in samples from the affected individual and is not influenced by CHX.

Article Snippet: Data derived from Oxford Nanopore long-read sequencing RT-PCR amplicons (exons 13–17) produced from RNA isolated following transactivation of SCN1A in HDFs derived from healthy control and affected individuals in the presence and absence of CHX.

Techniques: Expressing, Variant Assay, Derivative Assay, Sequencing, Reverse Transcription Polymerase Chain Reaction, Produced, Isolation, Control

Journal: American Journal of Human Genetics

Article Title: RNA variant assessment using transactivation and transdifferentiation

doi: 10.1016/j.ajhg.2024.06.018

Figure Lengend Snippet: Investigation of RNA variants in SMGs using transactivation of HDFs (A) Illustration of USH2A mRNA expression (red) in human adult tissues as reported by the Human Protein Atlas (HPA). (B) Diagram depicts the USH2A variant under investigation. Sashimi plots report USH2A mRNA splicing. Data derived from Oxford Nanopore long read sequencing of RT-PCR amplicons (exons 13–16) produced using RNA isolated following transactivation of USH2A in HDFs derived from healthy control and affected individuals in the presence and absence of cycloheximide (CHX). Arrows in the sashimi plots specify the reads coming from the alleles with pathogenic variant and allele with VUS as segregated by allelic phasing. (C) Illustration of SCN1A mRNA expression (red) in human adult tissues (HPA). (D) Diagram depicts the SCN1A variant under investigation. Sashimi plots report SCN1A mRNA splicing. Data derived from Oxford Nanopore long-read sequencing RT-PCR amplicons (exons 13–17) produced from RNA isolated following transactivation of SCN1A in HDFs derived from healthy control and affected individuals in the presence and absence of CHX. Arrows on the sashimi plot indicates the position of the pathogenic variant. (E) Illustration of DMD mRNA expression (red) in human adult tissues (HPA). (F) Diagram depicts the DMD variant under investigation. Graphs represent relative read depth of reported across DMD exons 2–5 as determined using long read sequencing of RT-PCR amplicons produced from RNA isolated following transactivation of DMD in HDFs derived from healthy control and affected individuals in the presence and absence of CHX. Note read depth is 1.8 times greater (∼double) in exons 3– and 4 only in samples from the affected individual and is not influenced by CHX.

Article Snippet: We compared PAK3 splicing in transactivated HDFs and human iPSC-derived neurons using Oxford Nanopore long-read sequencing of amplicons generated by PCR of PAK3 cDNA.

Techniques: Expressing, Variant Assay, Derivative Assay, Sequencing, Reverse Transcription Polymerase Chain Reaction, Produced, Isolation, Control

Journal: Genome Biology and Evolution

Article Title: Nanopore Amplicon Sequencing Reveals Molecular Convergence and Local Adaptation of Rhodopsin in Great Lakes Salmonids

doi: 10.1093/gbe/evaa237

Figure Lengend Snippet: Summary of steps for amplicon sequencing and bioinformatic analyses. Boxes on the left represent individual steps, color-coded based on their phase: red represents sample preparation, yellow represents nanopore sequencing, green represents sample demultiplexing, blue represents read mapping, and purple represents genotyping and analysis. Larger boxes to the right show additional information for each of the steps: the simplified mechanisms by which amplicons are generated and barcoded (top); frequency histogram with read length on the x-axis and number of reads in the y-axis (middle); and how reads are mapped to the reference genome (bottom).

Article Snippet: Long-read amplicon sequencing using the Oxford Nanopore Flongle is highly amenable to genotyping complex eukaryotes.

Techniques: Amplification, Sequencing, Sample Prep, Nanopore Sequencing, Generated

Journal: Scientific Reports

Article Title: Detection of single nucleotide and copy number variants in the Fabry disease-associated GLA gene using nanopore sequencing

doi: 10.1038/s41598-021-01749-7

Figure Lengend Snippet: Genotyping results based on ONT amplicon sequencing, and predicted consequence of the genetic variant on GLA .

Article Snippet: The purpose of our study is to validate the technique of Oxford Nanopore long amplicon sequencing of the GLA gene.

Techniques: Amplification, Sequencing, Variant Assay