Journal: GigaScience

Article Title: Sequencing smart: De novo sequencing and assembly approaches for a non-model mammal

doi: 10.1093/gigascience/giaa045

Figure Lengend Snippet: Information regarding the possible resolution for various de novo genome sequencing technologies

Article Snippet: The 10x Genomics Chromium library was assembled using the 10x Genomics Supernova software [ ], using default parameters.

Techniques: Sequencing

Journal: GigaScience

Article Title: Sequencing smart: De novo sequencing and assembly approaches for a non-model mammal

doi: 10.1093/gigascience/giaa045

Figure Lengend Snippet: Ten different assembly strategies using a variety of different data types: PCR-free Illumina short-read (“PCR-free”), long mate pair (“LMP”), 10x Genomics Chromium library (“10x”), and Bionano Genomics optical maps (“Bionano”). The blue-boxed assemblies all originate from the same PCR-free w2rap assembly (A1), and the black-boxed assemblies all originate from the same 10x Genomics Supernova assembly (A3). Information in paretheses refers to assembly software pipeline, and assembly numbers are annotated below each assembly.

Article Snippet: The 10x Genomics Chromium library was assembled using the 10x Genomics Supernova software [ ], using default parameters.

Techniques: Software

Journal: GigaScience

Article Title: Sequencing smart: De novo sequencing and assembly approaches for a non-model mammal

doi: 10.1093/gigascience/giaa045

Figure Lengend Snippet: Genome assembly statistics (for sequences >1 kb) for all assemblies

Article Snippet: The 10x Genomics Chromium library was assembled using the 10x Genomics Supernova software [ ], using default parameters.

Techniques:

Journal: GigaScience

Article Title: Sequencing smart: De novo sequencing and assembly approaches for a non-model mammal

doi: 10.1093/gigascience/giaa045

Figure Lengend Snippet: Comparison of the number of breaks introduced by REAPR for each of the technologies used to scaffold the w2rap-only assembly (A1)

Article Snippet: The 10x Genomics Chromium library was assembled using the 10x Genomics Supernova software [ ], using default parameters.

Techniques: Comparison

Journal: GigaScience

Article Title: Sequencing smart: De novo sequencing and assembly approaches for a non-model mammal

doi: 10.1093/gigascience/giaa045

Figure Lengend Snippet: Comparison of the number of breaks introduced by REAPR for each of the technologies used to scaffold the 10x assembly (A3)

Article Snippet: The 10x Genomics Chromium library was assembled using the 10x Genomics Supernova software [ ], using default parameters.

Techniques: Comparison

Journal: GigaScience

Article Title: Sequencing smart: De novo sequencing and assembly approaches for a non-model mammal

doi: 10.1093/gigascience/giaa045

Figure Lengend Snippet: Repeat content of assemblies

Article Snippet: The 10x Genomics Chromium library was assembled using the 10x Genomics Supernova software [ ], using default parameters.

Techniques:

Journal: GigaScience

Article Title: Sequencing smart: De novo sequencing and assembly approaches for a non-model mammal

doi: 10.1093/gigascience/giaa045

Figure Lengend Snippet: Cumulative z -scores of assemblies (solid black circles). Error bars represent the minimum and maximum cumulative z -score after removing each metric in turn and recalculating the z -score for each assembly. Wide error bars show assemblies that are strongly affected by a given metric. For example, the 10x + lmp + bionano assembly (A8) has a long lower-boundary error bar because it has an exceptionally high scaffold N50 z -score (double that of the next nearest ranking assembly) and hence omitting this metric results in the assembly scoring much lower.

Article Snippet: The 10x Genomics Chromium library was assembled using the 10x Genomics Supernova software [ ], using default parameters.

Techniques:

Journal: GigaScience

Article Title: Sequencing smart: De novo sequencing and assembly approaches for a non-model mammal

doi: 10.1093/gigascience/giaa045

Figure Lengend Snippet: REAPR statistics showing the percentage of error-free bases in the assembly, N50s before and after breaking at breakpoints, the percentage decrease in scaffold N50 after breaking, and the fragment coverage distribution (FCD) errors including errors across gaps

Article Snippet: The 10x Genomics Chromium library was assembled using the 10x Genomics Supernova software [ ], using default parameters.

Techniques:

Journal: Cells

Article Title: Identification and Characterization of ATOH7-Regulated Target Genes and Pathways in Human Neuroretinal Development

doi: 10.3390/cells13131142

Figure Lengend Snippet: Graphical summary of the study design. Human iPSCs were edited using a CRISPR/Cas9 approach. From the edited cell population, single cells were sorted and expanded in 96-well plates, followed by genotyping by Sanger sequencing. Selected clones were then differentiated to retinal organoids, which were then collected for different experiments. iPSC, induced pluripotent stem cell; WT, wildtype; KO, knockout; CUT&RUN-seq, cleavage under targets and release using nuclease sequencing; scRNA-seq, single-cell RNA sequencing; MUT-GFP, mutant reporter; WT-GFP, wildtype reporter.

Article Snippet: To gain better insights into the identities of retinal cells expressing ATOH7 and its target genes, we performed scRNA-seq on ATOH7 WT and ATOH7 KO week 7 whole-organoid dissociations using Chromium Fixed RNA Profiling (10X Genomics, Pleasanton, CA, USA).

Techniques: CRISPR, Sequencing, Clone Assay, Knock-Out, RNA Sequencing, Mutagenesis

Journal: Cells

Article Title: Identification and Characterization of ATOH7-Regulated Target Genes and Pathways in Human Neuroretinal Development

doi: 10.3390/cells13131142

Figure Lengend Snippet: Schematic representation of the iPSC cell lines used in the current study, which include isogenic WT and KO clones ( ATOH7 WT and ATOH7 KO ), as well as WT and mutant eGFP reporter lines ( ATOH7 WT-GFP and ATOH7 MUT-GFP ). iPSC, induced pluripotent stem cell; WT, wildtype; KO, knockout; UTR, untranslated region; CDS, coding sequence; T, c-Myc tag; eGFP, enhanced green fluorescent protein; P2A, porcine teschovirus-1 2A self-cleaving peptide; HA, homology arm; sgKO, single-guide RNA for ATOH7 knockout; PAM, protospacer-adjacent motif; MUT-GFP, mutant reporter; WT-GFP, wildtype reporter.

Article Snippet: To gain better insights into the identities of retinal cells expressing ATOH7 and its target genes, we performed scRNA-seq on ATOH7 WT and ATOH7 KO week 7 whole-organoid dissociations using Chromium Fixed RNA Profiling (10X Genomics, Pleasanton, CA, USA).

Techniques: Clone Assay, Mutagenesis, Knock-Out, Sequencing

Journal: Cells

Article Title: Identification and Characterization of ATOH7-Regulated Target Genes and Pathways in Human Neuroretinal Development

doi: 10.3390/cells13131142

Figure Lengend Snippet: Retinal organoid cell composition and cell type-specific genes regulated by ATOH7. Ten week 7 ATOH7 WT and ATOH7 KO retinal organoids were separately pooled, dissociated, and subsequently analyzed by hybridization-based PFA-fixed scRNA-seq. ( A ) UMAP of defined retinal clusters from integrated ATOH7 WT and ATOH7 KO organoids. The representative clusters are defined as naïve retinal progenitor cells ( nRPCs ), transient retinal progenitor cells ( tRPCs ), early retinal ganglion cells ( early RGCs ), late retinal ganglion cells ( late RGCs ), horizontal and amacrine cells ( H&As ), and photoreceptor cells ( PRs ). ( B ) Pseudotime trajectory analysis starting from TP53 -positive dividing retinal progenitor cells. ( C ) Separated UMAPs of cells derived from either ATOH7 WT or ATOH7 KO organoids. Percentages represent the proportion of cells in a given cluster compared to the total number of cells. Values in parentheses indicate the relative change in cell proportions compared to WT. ( D ) UMAP of re-clustered ATOH7 -positive cells derived from ATOH7 WT organoids. ( E ) ATOH7-targeted cell type marker genes, defined as scRNA-seq marker genes (AUC > 0.75) with a significant differential expression (adj. p -value < 0.01) between ATOH7 MUT-GFP and ATOH7 WT-GFP cells, based on reporter-enriched mRNA-seq, and at least one binding site discovered by CUT&RUN-seq, where the annotation is defined as either enhancer (>5 kb from TSS) or promoter (±5 kb from TSS). AUC, area under curve; CUT&RUN-seq, cleavage under targets and release using nuclease sequencing; KO, knockout; PFA, paraformaldehyde; scRNA-seq, single-cell RNA sequencing; TSS, transcription start site; UMAP, uniform manifold approximation and projection; DEGs, differentially expressed genes between MUT-GFP and WT-GFP; MUT-GFP, mutant reporter; WT-GFP, wildtype reporter; WT, wildtype.

Article Snippet: To gain better insights into the identities of retinal cells expressing ATOH7 and its target genes, we performed scRNA-seq on ATOH7 WT and ATOH7 KO week 7 whole-organoid dissociations using Chromium Fixed RNA Profiling (10X Genomics, Pleasanton, CA, USA).

Techniques: Hybridization, Derivative Assay, Marker, Quantitative Proteomics, Binding Assay, Sequencing, Knock-Out, RNA Sequencing, Mutagenesis

Journal: Cells

Article Title: Identification and Characterization of ATOH7-Regulated Target Genes and Pathways in Human Neuroretinal Development

doi: 10.3390/cells13131142

Figure Lengend Snippet: Axon guidance and Notch signaling are enriched amongst ATOH7 target DEGs. Identified ATOH7 target genes and ATOH7 -associated DEGs in eGFP-enriched reporter cells derived from week 7 retinal organoids were overlapped to identify differentially expressed ATOH7 target genes (ATOH7 target DEGs). ( A ) Venn diagram showing the identification of ATOH7 target DEGs. ( B ) Number of regulated genes per cell type amongst ATOH7 target DEGs, with cell-type specificity defined by scRNA-seq. The representative cell clusters are defined as naïve retinal progenitor cells ( nRPCs ), transient retinal progenitor cells ( tRPCs ), early retinal ganglion cells ( early RGCs ), late retinal ganglion cells ( late RGCs ), horizontal and amacrine cells ( H&As ), and photoreceptor cells ( PRs ). ( C ) Enrichment of GO terms amongst the 469 ATOH7 target DEGs compared to all annotated genes. The analysis was performed in g:Profiler and reduced by REVIGO, showing the top five most significant terms for biological processes ( GO:BP ), cellular compartments ( GO:CC ), and molecular function ( GO:MF ) for ATOH7 target DEGs. Additionally, the enrichment of biological pathway terms from combined KEGG, REACTOME, and WikiPathways databases was performed against all known human genes, showing top 15 terms according to adjusted p -value. Asterisks (*) demark significance (adj. p -value < 0.05) when including annotated genes only. Multiple correction testing was performed using g:SCS. ( D ) Selection of ATOH7 target DEGs intersecting with top significantly enriched biological pathways. Similar terms from different databases were grouped together. “Nervous System Development” (R-HSA-9675108) and “Axon Guidance” (R-HSA-422475) were grouped together due to an almost complete overlap (98%) of intersecting genes. Heatmaps of annotated genes present relative expression per cell type, based on scRNA-seq data of ATOH7 -positive cells in ATOH7 WT organoids, differential expression between ATOH7 MUT-GFP and ATOH7 WT-GFP cells, based on reporter-enriched mRNA-seq, and the number of annotated ATOH7-binding loci identified by CUT&RUN-seq, where the annotation is defined as either enhancer (>5 kb from TSS) or promoter (±5 kb from TSS). ( E ) Expression distribution of selected genes in cell type-specific scRNA-seq clusters. Violin plots show raw expression distribution for cells originating from ATOH7 WT organoids, ATOH7 KO organoids, and from the ATOH7 -expressing ( ATOH7 + ) cells, re-clustered from the ATOH7 WT organoids. Each expressing cell is marked by a black point. CUT&RUN-seq, cleavage under targets and release using nuclease sequencing; EVR, exudative vitreoretinopathy; g:SCS, g:Profiler set counts and sizes; GO, gene ontology; KO, knockout; REVIGO, reduce visualize gene ontology; PHPV, persistent hyperplastic primary vitreous; scRNA-seq, single-cell RNA sequencing; DEGs, differentially expressed genes between MUT-GFP and WT-GFP; MUT-GFP, mutant reporter; WT-GFP, wildtype reporter; TSS, transcription start site; WT, wildtype.

Article Snippet: To gain better insights into the identities of retinal cells expressing ATOH7 and its target genes, we performed scRNA-seq on ATOH7 WT and ATOH7 KO week 7 whole-organoid dissociations using Chromium Fixed RNA Profiling (10X Genomics, Pleasanton, CA, USA).

Techniques: Derivative Assay, Selection, Expressing, Quantitative Proteomics, Binding Assay, Sequencing, Knock-Out, RNA Sequencing, Mutagenesis

Journal: Cells

Article Title: Identification and Characterization of ATOH7-Regulated Target Genes and Pathways in Human Neuroretinal Development

doi: 10.3390/cells13131142

Figure Lengend Snippet: ATOH7 regulates genes encoding Wnt effectors and other secreted proteins. ( A ) Selection of ATOH7 reporter DEGs (log 2 FC > ±2, FDR < 0.01) encoding extracellular/secreted proteins. Heatmap of annotated genes present relative expression per cell type, based on scRNA-seq data, differential expression between ATOH7 MUT-GFP and ATOH7 WT-GFP cells, based on reporter-enriched mRNA-seq, and the number of annotated ATOH7-binding loci identified by CUT&RUN-seq, where the annotation is defined as either enhancer (>5 kb from TSS) or promoter (±5 kb from TSS). The representative cell clusters are defined as naïve retinal progenitor cells ( nRPCs ), transient retinal progenitor cells ( tRPCs ), early retinal ganglion cells ( early RGCs ), late retinal ganglion cells ( late RGCs ), horizontal and amacrine cells ( H&As ), and photoreceptor cells ( PRs ). ( B ) Expression distribution of selected genes encoding secreted proteins in cell type-specific scRNA-seq clusters. Violin plots show raw expression distribution in cells originating from ATOH7 WT organoids, ATOH7 KO organoids, and from the ATOH7 expressing ( ATOH7 + ) cells, re-clustered from the ATOH7 WT organoids. Each expressing cell is marked by a black dot. ( C ) Heatmap including the selection of ATOH7 reporter DEGs (log 2 FC > ±2, FDR < 0.01) associated with PHPV/EVR in patients ( LRP5 , NDP , KIF11 , and RCBTB1 ) and/or in vivo ( LRP5 , NDP , KIF11 , EFNA5 , EFNB2 , and NEO1 ) or in silico ( SMAD2 and, MYCN ) according to a literature search [ , , , , , , , , , , , , , , , , , , , , , , , , , , ]. ( D ) Expression distribution of genes encoding NDP and NDP -associated receptors in cell type-specific scRNA-seq clusters. CUT&RUN-seq , cleavage under targets and release using nuclease sequencing; EVR , exudative vitreoretinopathy; KO , knockout; PHPV , persistent hyperplastic primary vitreous; scRNA-seq , single-cell RNA sequencing; DEGs , differentially expressed genes between MUT-GFP and WT-GFP; MUT-GFP , mutant reporter; WT-GFP , wildtype reporter; TSS , transcription start site; WT , wildtype.

Article Snippet: To gain better insights into the identities of retinal cells expressing ATOH7 and its target genes, we performed scRNA-seq on ATOH7 WT and ATOH7 KO week 7 whole-organoid dissociations using Chromium Fixed RNA Profiling (10X Genomics, Pleasanton, CA, USA).

Techniques: Selection, Expressing, Quantitative Proteomics, Binding Assay, In Vivo, In Silico, Sequencing, Knock-Out, RNA Sequencing, Mutagenesis

Journal: Nature Communications

Article Title: Integrative genotyping of cancer and immune phenotypes by long-read sequencing

doi: 10.1038/s41467-023-44137-7

Figure Lengend Snippet: a Intermediate single-cell cDNA libraries of cancer specimens are used for targeted amplification of transcripts carrying natural barcodes. By sequencing long amplicons on the Oxford Nanopore platform multiple genetic barcodes such as somatic nuclear mutations can be detected from the same amplification product. Applications for long-read sequencing include the read-out of T cell receptor (TCR) or CAR sequences, somatic nuclear and mitochondrial DNA mutations, fusion transcripts, and alternative splicing events (gene isoforms). The genotyping information from long-read sequencing is integrated with the gene expression data from short-read sequencing. b Amplicons are generated using a 3-step PCR. In the first PCR template-switch oligo (TSO) artifacts are removed with generic amplification of cDNA using a biotinylated 3′ primer and streptavidin purification. In the second PCR, gene-specific biotinylated 3′ primers are used to amplify loci of interest. After a second streptavidin purification, target genes are amplified with nested gene-specific 3′ primers to provide sufficient material for sequencing. c Overview of the nanoranger workflow. Multimer reads are deconcatenated by identifying transcripts with alignment against a reference transcriptome (1). After extraction of subreads (2), cell barcodes are identified (3) and TCR information is processed or transcripts are genome-aligned (4) for downstream genotyping. d Examples of gene coverage with (black) and without (gray) removal of TSO artifacts. The blue line indicates the primer binding site and the red ribbon shows locations of mutations used for lineage tracking in AML detected with each primer set. e Benchmarking of multimer demultiplexing using nanoranger and longbow on artificially generated multimers using the ISO-MAS-seq protocol. Of note, nanoranger deconcatenates multimer reads agnostic of adapters between transcripts, while longbow is optimized for known adapter sequences.

Article Snippet: We re-sequenced a TCR cDNA library generated from melanoma-infiltrating T cells (originally processed with the 10x Genomics (V)DJ kit) using both the Illumina and ONT sequencing platforms (Fig. , Supplementary Fig. , Supplementary Table ).

Techniques: Amplification, Sequencing, Alternative Splicing, Gene Expression, Generated, Purification, Extraction, Binding Assay

Journal: Nature Communications

Article Title: Integrative genotyping of cancer and immune phenotypes by long-read sequencing

doi: 10.1038/s41467-023-44137-7

Figure Lengend Snippet: a Tumor-infiltrating T cells from one melanoma case (patient C described in Oliveira et al., Nature 2021 ) were isolated and used for single cell sequencing. After generation of enriched T cell receptor (TCR) libraries, they were either fragmented and sequenced with Illumina (left) or directly sequenced without fragmentation with Oxford Nanopore (ONT) (right). b Comparison of reads obtained with Illumina and ONT ( nanoranger ) per cell barcode shown for TCRα (light) and TCRβ (dark) sequences (left) or number of cells obtained with Illumina and ONT with a particular CDR3α (light) or CDR3β (dark) (right). The rectangle indicates unproductive CDR3s filtered by cellranger. c UMAP representation of cell types (top left), cells with detectable TCR (top right), expression of CD14 and CD3E (bottom). The bar plot demonstrates the number of TCR sequences obtained by Illumina and ONT sequencing. d Overview mixing experiment with Kasumi-1 (acute myeloid leukemia, AML) and K562 (chronic myeloid leukemia, CML) cells. Kasumi-1 contain a homozygous TP53 R248G mutation and the RUNX1::RUNX1T1 fusion gene. K562 express only one TP53 allele with a truncating frameshift mutation ( TP53 Q136fs ) and BCR::ABL1 . Both TP53 mutations are detected with the same primer. e Absolute number and percentage of genotyped Kasumi-1 (red) and K562 cells (yellow) for the TP53 R248G mutation (left) and percentage of cells carrying either TP53 R248G or TP53 Q136fs (right). f Expression of TP53 and percentage of cells with detectable TP53 transcripts (left). Number of cells genotyped as function of the number of TP53 reads shown for Kasumi-1 (red) and K562 (yellow) in a downsampling experiment (right). g Percentage of genotyped cells for BCR::ABL1 and RUNX1::RUNX1T1 (left). Percentage of cells with BCR::ABL1 and RUNX1::RUNX1T1 shown for Kasumi-1 (red) and K562 (yellow) (right). h Coverage across all detectable genes from a 10x Genomics cDNA library after removal of TSO artifacts as function of transcript length.

Article Snippet: We re-sequenced a TCR cDNA library generated from melanoma-infiltrating T cells (originally processed with the 10x Genomics (V)DJ kit) using both the Illumina and ONT sequencing platforms (Fig. , Supplementary Fig. , Supplementary Table ).

Techniques: Isolation, Sequencing, Comparison, Expressing, Mutagenesis, cDNA Library Assay

Journal: Nature Communications

Article Title: Integrative genotyping of cancer and immune phenotypes by long-read sequencing

doi: 10.1038/s41467-023-44137-7

Figure Lengend Snippet: a Experimental workflow of comparison between nanoranger and genotyping of transcriptomes (GoT). A pretreatment bone marrow sample of AML1022 at relapse after allogeneic hematopoietic stem cell transplantation (HSCT) was used for single cell cDNA library preparation according to the standard 10x Genomics 5′ gene expression protocol and following the modified 5′ GoT protocol with in-droplet inclusion of gene-specific reverse transcriptase primers. Both cDNAs were taken forward for sequencing with the standard nanoranger protocol (orange), GoT using Illumina sequencing (black) and GoT using Oxford Nanopore sequencing (blue). b , c Number of cells genotyped with each experimental condition (b) and percentage of genotyped cells across hematopoietic differentiation states ( c ). d Comparison of apparent single cell variant allele frequencies (VAFs) for SF3B1 K700E in donor- versus recipient-derived cells to demonstrate specificity of genotyping with each experimental condition. e Comparison of cell barcodes identified with each condition. The venn diagrams demonstrate the number of cell barcodes that are uniquely identified or shared across experimental conditions. To enable direct comparison of captured cell barcodes, the cDNA for the GoT condition was used as input for nanoranger . f Minimal read length versus number of reads for cell barcodes identified with GoT on Illumina and ONT (black) versus those identified only with GoT on ONT (blue), demonstrating the preferential sequencing of shorter fragments with Illumina sequencing.

Article Snippet: We re-sequenced a TCR cDNA library generated from melanoma-infiltrating T cells (originally processed with the 10x Genomics (V)DJ kit) using both the Illumina and ONT sequencing platforms (Fig. , Supplementary Fig. , Supplementary Table ).

Techniques: Comparison, Transplantation Assay, cDNA Library Assay, Gene Expression, Modification, Reverse Transcription, Sequencing, Illumina Sequencing, Nanopore Sequencing, Variant Assay, Derivative Assay

Journal: Nature Communications

Article Title: Integrative genotyping of cancer and immune phenotypes by long-read sequencing

doi: 10.1038/s41467-023-44137-7

Figure Lengend Snippet: a Co-existence of two subclones in de-novo AML 1. Detection of the somatic nuclear mutations NPM1 W287fs (clone 1) and NPM1 W288fs (clone 2) demonstrates co-existence of two AML clones that differ in the presence of FLT3-ITD and loss of heterozygosity on chromosome 13 ( loh(13) ) as well as several mitochondrial DNA mutations. b Differential analysis of bulk mitochondrial DNA heteroplasmy between clone 1 and clone 2 in de-novo AML 1. c Heatmap demonstrating molecular features of clone 1 and clone 2 in 525 cells of de-novo AML 1. d , e Differential gene expression analysis (DGEA) between GMP-like cells of clone 1 and clone 2. f Comparison of BCR::ABL1 amplicons in two Philadelphia + (Ph + ) acute lymphoblastic leukemia (ALL) cases, in K562 cells and Ph + chronic myeloid leukemia (CML). In all 4 cases the same ABL1 -specific primer was used. The p190 variant in ALL produces a shorter fusion transcript than the p210 variant in CML making it more detectable with targeted long-read sequencing from 10x Genomics cDNA libraries. g Identification of BCR::ABL1 + cells in ALL bone marrow. UMAP plots show cell type annotation (left), samples (middle) and detection of BCR::ABL1 transcripts (right) in ALL1 (black) and ALL2 (red). h BCR::ABL1 + cells in bone marrow of ALL1 and ALL2 (left) and cells with detectable CNV changes in re-analyzed ALL datasets from refs. and (right), mapped to a healthy bone marrow reference.

Article Snippet: We re-sequenced a TCR cDNA library generated from melanoma-infiltrating T cells (originally processed with the 10x Genomics (V)DJ kit) using both the Illumina and ONT sequencing platforms (Fig. , Supplementary Fig. , Supplementary Table ).

Techniques: Clone Assay, Gene Expression, Comparison, Variant Assay, Sequencing

Journal: Nature Communications

Article Title: Integrative genotyping of cancer and immune phenotypes by long-read sequencing

doi: 10.1038/s41467-023-44137-7

Figure Lengend Snippet: a Targeted amplification of PTPRC to detect differential splicing of exon 4 which determines expression of CD45RA (exon 4 expressed) versus CD45RO (exon 4 not expressed) (top). Targeted amplification dramatically increases coverage of PTPRC (red) compared to whole-transcriptome (WT) amplified cDNA (gray), both sequenced on the Oxford Nanopore platform. b UMAP representation of tumor-infiltrating T cells (TILs) and circulating T cells from melanoma Patient C (Oliveira et al., Nature 2021 ) . The top row shows expression of PTPRC exon 4 (CD45RA) and the bottom row shows CD45RA protein expression measured by CITE-seq. c Expression of PTPRC (exon 4) (left) and CD45RA measured by CITE-seq (right) across T cell subsets in bone marrow of AML1007 before infusion of ipilimumab (baseline) and after 1 or 4 cycles of ipilimumab. d Targeted amplification of CTLA-4 to detect exon 3 which discriminates the soluble (exon 3 absent) and membranous (exon 3 present) isoforms (top). Knee plot demonstrating the high degree of enrichment of CTLA-4 transcripts with targeted long-read sequencing (red) versus whole-transcriptome long-read sequencing (gray) (bottom). e Expression of soluble (black) and membranous (yellow) isoforms of CTLA-4 across AML3005, tumor-infiltrating T cells of Patient C, and a CAR T cell infusion product (top). Expression level of CTLA-4 as measured by short-read sequencing indicates specific detection with the targeted approach (bottom). f Distribution of percentage of reads with membranous CTLA-4 across eight different T cell single-cell cDNA libraries for a total of 4786 cells. Patients C and D were previously described by ref. . CAR IP - CAR T cell infusion product.

Article Snippet: We re-sequenced a TCR cDNA library generated from melanoma-infiltrating T cells (originally processed with the 10x Genomics (V)DJ kit) using both the Illumina and ONT sequencing platforms (Fig. , Supplementary Fig. , Supplementary Table ).

Techniques: Amplification, Expressing, Sequencing