Journal: bioRxiv

Article Title: Pushing the limits of single molecule transcript sequencing to uncover the largest disease-associated transcript isoforms in the human neural retina

doi: 10.1101/2024.09.10.612265

Figure Lengend Snippet: The figure illustrates the sequencing workflows and subsequent analysis performed on RNA extracted from three human neural retina samples. The workflows included PacBio long-read mRNA Iso-Seq using both the standard and an optimized long transcript workflow. The analysis was carried out in three distinct datasets: Dataset 1 comprised the standard workflow samples analyzed with IsoQuant, Dataset 2 involved a combined analysis of the standard and optimized long transcript workflows, and Dataset 3 focused solely on data from the long transcript workflow. Additionally, an indirect targeted enrichment of transcripts for the USH2A and ADGRV1 genes was achieved using the Samplix Xdrop System, followed by PacBio long-read sequencing and cDNA analysis. Additionally, the sequenced reads were manually curated using BAM files in the Integrative Genomics Viewer. Integrating data from the three sequencing workflows and combining algorithmic analysis with manual curation enabled a thorough characterization of the Usher syndrome-associated transcript isoforms present in the human neural retina. Validation of selected transcripts and events was performed using an independent Oxford Nanopore Technology (ONT) long-read sequencing dataset of three independent retina samples.

Article Snippet: The sample mix, prepared using the “Setup of droplet PCR” and “Positive control DNA enrichment droplet PCR” sections of the Samplix “Targeted enrichment of DNA with Xdrop or Xdrop Sort” protocol version 2.0, was loaded onto the Xdrop DE20 cartridge.

Techniques: Sequencing

Journal: bioRxiv

Article Title: Pushing the limits of single molecule transcript sequencing to uncover the largest disease-associated transcript isoforms in the human neural retina

doi: 10.1101/2024.09.10.612265

Figure Lengend Snippet: The GENCODE reference transcript is depicted at the top in green, followed by the proposed USH2A transcript isoforms and events based on manual curation of BAM-files using the Integrative Genomics Viewer (IGV) in red, and proposed transcript isoforms following the Samplix Xdrop targeted enrichment in orange. The light green, red and orange colors indicate the untranslated regions (UTR) and the dark green, red and orange colors indicate the open reading frame (ORF) of each transcript. Differences between the proposed transcript isoforms and the GENCODE reference transcript are highlighted in grey boxes. The overview of sporadic incorporation of cryptic exons indicates the presence of PE8 and PE20 as previously described by Reurink et al . (2023) . Additionally, locatons where cryptic exons are occasionally incorporated at sites that are not yet noted in literature are highlighted by the overlaying black arrows in the overview.

Article Snippet: The sample mix, prepared using the “Setup of droplet PCR” and “Positive control DNA enrichment droplet PCR” sections of the Samplix “Targeted enrichment of DNA with Xdrop or Xdrop Sort” protocol version 2.0, was loaded onto the Xdrop DE20 cartridge.

Techniques:

Journal: bioRxiv

Article Title: Pushing the limits of single molecule transcript sequencing to uncover the largest disease-associated transcript isoforms in the human neural retina

doi: 10.1101/2024.09.10.612265

Figure Lengend Snippet: The GENCODE reference transcript is depicted at the top in green, followed by the ADGRV1 proposed transcript isoforms and events based on manual curation of BAM-files using the Integrative Genomics Viewer (IGV) in red, and proposed transcript isoforms following the Samplix Xdrop targeted enrichment in orange. The light green, red and orange colors indicate the untranslated regions (UTR) and the dark green, red and orange colors indicate the open reading frame (ORF) of each transcript. Differences between the proposed transcript isoforms and the GENCODE reference transcript are highlighted in grey box.

Article Snippet: The sample mix, prepared using the “Setup of droplet PCR” and “Positive control DNA enrichment droplet PCR” sections of the Samplix “Targeted enrichment of DNA with Xdrop or Xdrop Sort” protocol version 2.0, was loaded onto the Xdrop DE20 cartridge.

Techniques: