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Oxford Nanopore sequence dna rna molecules
Sequence Dna Rna Molecules, supplied by Oxford Nanopore, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Sequencing:

Article Title: Tools for Genomic and Transcriptomic Analysis of Microbes at Single-Cell Level
Article Snippet: .. Recently, new sequencing platforms such as true single molecule sequencing (tSMS, Helicos, now SeqLL), SMRT sequencing (PacBio), and nanopore sequencing (Oxford Nanopore) could sequence DNA/RNA molecules at single-molecule level and prove to be possible to sequence DNA/RNA molecules directly from bulk-cells without pre-amplification ( ; ; ; ). .. Although directly sequencing a single cell without pre-amplification is still challenging, further innovation of these new technologies and sequencing platforms could eventually make it possible for single-cell analysis without any amplification.

Nanopore Sequencing:

Article Title: Tools for Genomic and Transcriptomic Analysis of Microbes at Single-Cell Level
Article Snippet: .. Recently, new sequencing platforms such as true single molecule sequencing (tSMS, Helicos, now SeqLL), SMRT sequencing (PacBio), and nanopore sequencing (Oxford Nanopore) could sequence DNA/RNA molecules at single-molecule level and prove to be possible to sequence DNA/RNA molecules directly from bulk-cells without pre-amplification ( ; ; ; ). .. Although directly sequencing a single cell without pre-amplification is still challenging, further innovation of these new technologies and sequencing platforms could eventually make it possible for single-cell analysis without any amplification.

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  • 84
    Oxford Nanopore rna molecule umi
    UMIs enable identification of PCR artefacts and sequencing error correction. (a) UMIs allow identification and elimination of reads originating from chimeric cDNA generated during PCR amplification. Chimeric cDNAs are mainly generated in later PCR cycles when cDNA concentration becomes higher. This results in a small fraction of reads ( , n : PCR cycle where chimera is generated) with aberrant exon layout. Those inconsistent reads are discarded, and the remaining reads are used to define the consensus cDNA sequence for the <t>UMI.</t> (b, c) UMIs enable sequencing error correction. Generation of consensus sequences for all reads associated with each UMI allows to obtain high accuracy sequences for each UMI <t>(RNA</t> molecule) despite the low accuracy of each Nanopore read. (c) the box plots indicate the accuracy of the cDNA consensus sequences (% identity to the reference genome) for different UMI sequencing depths. Boxes represent the 25% quantile to 75% quantile range, upper and lower edges of notches are median +/− 1.58 * IQR / sqrt(n) (IQR : inter quantile range, n: sample size).
    Rna Molecule Umi, supplied by Oxford Nanopore, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Oxford Nanopore native rna molecules
    Direct <t>RNA</t> barcoding and demultiplexing (A) Overview of Oxford <t>Nanopore</t> sample preparation protocol for native RNA sequencing. (B) Adaptation of (A) to include custom DNA barcodes. (C) Barcode segmentation and transformation, where the electric current associated with a barcode adapter (highlighted in red) is extracted and converted into an image using GASF transformation. (D) Deep learning is used to classify the segmented and GASF-transformed squiggle signals into their corresponding bins, without the need of base-calling the underlying sequence. The convolution architecture of the final residual neural network classifier (ResNet-20) described in this work: FC = Fully Connected layer.
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    80
    Oxford Nanopore single molecule direct rna sequencing
    <t>Nanopore</t> Sequencing. (A) Schematic of the library preparation procedure for Nanopore direct <t>RNA</t> sequencing. PolyA RNA is enriched using oligo-dT primers and a reverse transcription (RT) adaptor is ligated. After second-strand synthesis, the sequencing adapter RMX, which is preloaded with motor protein and tether protein, is then ligated. (B) Schematic of Nanopore direct RNA sequencing. The motor protein feeds the RNA molecule through the nanopore in the 3′–5′ direction. The five bases passing through a nanopore cause a characteristic disruption in the current which is stored as raw signal. (C) A current trace (squiggle plot) showing the raw signal generated by nanopore sequencing of a single mRNA molecule. Leader and adapter sequences are shaded yellow and pink, the polyA tail is shaded green, and the mRNA body is shaded orange. The inset (top right) illustrates how the nucleotide sequence is inferred from the raw current trace originating from a sliding window of five nucleotides (k-mer). Machine-learning algorithms are then used to calculate the probability that a signal corresponds to a given k-mer, thus inferring the nucleotide sequence from the calculated probabilities. (D) The two features recorded by Oxford Nanopore Technologies (ONT) sequencers are the current signal (in arbitrary units, AU) and the time that a given k-mer takes to transverse the pore (signal length, retention time or 'dwell'). The scatter plot depicts the distribution of mean current and signal length for 100 reads each in a different sequence context of the unmodified k-mer CACCC (blue) and the modified k-mer CAm 5 CCC (orange, identified by parallel bisulfite sequencing, where m 5 C is 5-methylcytosine). Note that, despite an identical k-mer, the signal varies as a result of different measurements and intrinsic noise in different reads, and possibly also by the different surrounding sequence of a given k-mer. This variability can be represented as a signal density plot for each k-mer, depicted in the top-right inset (density distribution for raw current signal). RNA modifications can affect raw current reads as well as signal length, resulting in a shift in signal distributions (e.g., divergence between blue and orange). However, these signal shifts can be modest, as shown by the largely overlapping density plots for CACCC and CAm 5 CCC, making accurate prediction of modified bases a computational challenge. Plots were generated with Sequoia, an interactive visual analytics platform for interpretation and feature extraction from ONT sequencing datasets [ 138 ].
    Single Molecule Direct Rna Sequencing, supplied by Oxford Nanopore, used in various techniques. Bioz Stars score: 80/100, based on 0 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Oxford Nanopore dna molecule
    Oxford <t>Nanopore</t> Technology read length distribution and CHEF electrophoresis of <t>DNA</t> from mitochondrial enriched T. gondii cell fractions. (A) Profile of read counts plotted by length for mtDNA (top) and nuclear (bottom). Species and strains are as indicated in the legend. (B) Southern of a Contour-clamped homogeneous electric field gel electrophoresis of T. gondii total DNA (Lane 1) and mitochondrial-enriched DNA (Lane 2) probed with a 1012 bp section of the cob gene. DNA ladder is as indicated. A plot of the ME49 and Nc-1 nuclear reads is located at the bottom of Table S3 since they could not be plotted here due to difference in read count scale.
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    UMIs enable identification of PCR artefacts and sequencing error correction. (a) UMIs allow identification and elimination of reads originating from chimeric cDNA generated during PCR amplification. Chimeric cDNAs are mainly generated in later PCR cycles when cDNA concentration becomes higher. This results in a small fraction of reads ( , n : PCR cycle where chimera is generated) with aberrant exon layout. Those inconsistent reads are discarded, and the remaining reads are used to define the consensus cDNA sequence for the UMI. (b, c) UMIs enable sequencing error correction. Generation of consensus sequences for all reads associated with each UMI allows to obtain high accuracy sequences for each UMI (RNA molecule) despite the low accuracy of each Nanopore read. (c) the box plots indicate the accuracy of the cDNA consensus sequences (% identity to the reference genome) for different UMI sequencing depths. Boxes represent the 25% quantile to 75% quantile range, upper and lower edges of notches are median +/− 1.58 * IQR / sqrt(n) (IQR : inter quantile range, n: sample size).

    Journal: bioRxiv

    Article Title: High throughput, error corrected Nanopore single cell transcriptome sequencing

    doi: 10.1101/831495

    Figure Lengend Snippet: UMIs enable identification of PCR artefacts and sequencing error correction. (a) UMIs allow identification and elimination of reads originating from chimeric cDNA generated during PCR amplification. Chimeric cDNAs are mainly generated in later PCR cycles when cDNA concentration becomes higher. This results in a small fraction of reads ( , n : PCR cycle where chimera is generated) with aberrant exon layout. Those inconsistent reads are discarded, and the remaining reads are used to define the consensus cDNA sequence for the UMI. (b, c) UMIs enable sequencing error correction. Generation of consensus sequences for all reads associated with each UMI allows to obtain high accuracy sequences for each UMI (RNA molecule) despite the low accuracy of each Nanopore read. (c) the box plots indicate the accuracy of the cDNA consensus sequences (% identity to the reference genome) for different UMI sequencing depths. Boxes represent the 25% quantile to 75% quantile range, upper and lower edges of notches are median +/− 1.58 * IQR / sqrt(n) (IQR : inter quantile range, n: sample size).

    Article Snippet: Grouping of reads that corresponded to the same RNA molecule (UMI) allowed correction of Nanopore sequencing errors, generation of consensus sequence for each RNA molecule and identification of such sequence heterogeneity within single cells ( ).

    Techniques: Polymerase Chain Reaction, Sequencing, Generated, Amplification, Concentration Assay

    Direct RNA barcoding and demultiplexing (A) Overview of Oxford Nanopore sample preparation protocol for native RNA sequencing. (B) Adaptation of (A) to include custom DNA barcodes. (C) Barcode segmentation and transformation, where the electric current associated with a barcode adapter (highlighted in red) is extracted and converted into an image using GASF transformation. (D) Deep learning is used to classify the segmented and GASF-transformed squiggle signals into their corresponding bins, without the need of base-calling the underlying sequence. The convolution architecture of the final residual neural network classifier (ResNet-20) described in this work: FC = Fully Connected layer.

    Journal: bioRxiv

    Article Title: Barcoding and demultiplexing Oxford Nanopore native RNA sequencing reads with deep residual learning

    doi: 10.1101/864322

    Figure Lengend Snippet: Direct RNA barcoding and demultiplexing (A) Overview of Oxford Nanopore sample preparation protocol for native RNA sequencing. (B) Adaptation of (A) to include custom DNA barcodes. (C) Barcode segmentation and transformation, where the electric current associated with a barcode adapter (highlighted in red) is extracted and converted into an image using GASF transformation. (D) Deep learning is used to classify the segmented and GASF-transformed squiggle signals into their corresponding bins, without the need of base-calling the underlying sequence. The convolution architecture of the final residual neural network classifier (ResNet-20) described in this work: FC = Fully Connected layer.

    Article Snippet: Nanopore sequencing has enabled sequencing of native RNA molecules without conversion to cDNA, thus opening the gates to a new era for the unbiased study of RNA biology.

    Techniques: Sample Prep, RNA Sequencing Assay, Transformation Assay, Sequencing

    Nanopore Sequencing. (A) Schematic of the library preparation procedure for Nanopore direct RNA sequencing. PolyA RNA is enriched using oligo-dT primers and a reverse transcription (RT) adaptor is ligated. After second-strand synthesis, the sequencing adapter RMX, which is preloaded with motor protein and tether protein, is then ligated. (B) Schematic of Nanopore direct RNA sequencing. The motor protein feeds the RNA molecule through the nanopore in the 3′–5′ direction. The five bases passing through a nanopore cause a characteristic disruption in the current which is stored as raw signal. (C) A current trace (squiggle plot) showing the raw signal generated by nanopore sequencing of a single mRNA molecule. Leader and adapter sequences are shaded yellow and pink, the polyA tail is shaded green, and the mRNA body is shaded orange. The inset (top right) illustrates how the nucleotide sequence is inferred from the raw current trace originating from a sliding window of five nucleotides (k-mer). Machine-learning algorithms are then used to calculate the probability that a signal corresponds to a given k-mer, thus inferring the nucleotide sequence from the calculated probabilities. (D) The two features recorded by Oxford Nanopore Technologies (ONT) sequencers are the current signal (in arbitrary units, AU) and the time that a given k-mer takes to transverse the pore (signal length, retention time or 'dwell'). The scatter plot depicts the distribution of mean current and signal length for 100 reads each in a different sequence context of the unmodified k-mer CACCC (blue) and the modified k-mer CAm 5 CCC (orange, identified by parallel bisulfite sequencing, where m 5 C is 5-methylcytosine). Note that, despite an identical k-mer, the signal varies as a result of different measurements and intrinsic noise in different reads, and possibly also by the different surrounding sequence of a given k-mer. This variability can be represented as a signal density plot for each k-mer, depicted in the top-right inset (density distribution for raw current signal). RNA modifications can affect raw current reads as well as signal length, resulting in a shift in signal distributions (e.g., divergence between blue and orange). However, these signal shifts can be modest, as shown by the largely overlapping density plots for CACCC and CAm 5 CCC, making accurate prediction of modified bases a computational challenge. Plots were generated with Sequoia, an interactive visual analytics platform for interpretation and feature extraction from ONT sequencing datasets [ 138 ].

    Journal: Trends in Biotechnology

    Article Title: New Twists in Detecting mRNA Modification Dynamics

    doi: 10.1016/j.tibtech.2020.06.002

    Figure Lengend Snippet: Nanopore Sequencing. (A) Schematic of the library preparation procedure for Nanopore direct RNA sequencing. PolyA RNA is enriched using oligo-dT primers and a reverse transcription (RT) adaptor is ligated. After second-strand synthesis, the sequencing adapter RMX, which is preloaded with motor protein and tether protein, is then ligated. (B) Schematic of Nanopore direct RNA sequencing. The motor protein feeds the RNA molecule through the nanopore in the 3′–5′ direction. The five bases passing through a nanopore cause a characteristic disruption in the current which is stored as raw signal. (C) A current trace (squiggle plot) showing the raw signal generated by nanopore sequencing of a single mRNA molecule. Leader and adapter sequences are shaded yellow and pink, the polyA tail is shaded green, and the mRNA body is shaded orange. The inset (top right) illustrates how the nucleotide sequence is inferred from the raw current trace originating from a sliding window of five nucleotides (k-mer). Machine-learning algorithms are then used to calculate the probability that a signal corresponds to a given k-mer, thus inferring the nucleotide sequence from the calculated probabilities. (D) The two features recorded by Oxford Nanopore Technologies (ONT) sequencers are the current signal (in arbitrary units, AU) and the time that a given k-mer takes to transverse the pore (signal length, retention time or 'dwell'). The scatter plot depicts the distribution of mean current and signal length for 100 reads each in a different sequence context of the unmodified k-mer CACCC (blue) and the modified k-mer CAm 5 CCC (orange, identified by parallel bisulfite sequencing, where m 5 C is 5-methylcytosine). Note that, despite an identical k-mer, the signal varies as a result of different measurements and intrinsic noise in different reads, and possibly also by the different surrounding sequence of a given k-mer. This variability can be represented as a signal density plot for each k-mer, depicted in the top-right inset (density distribution for raw current signal). RNA modifications can affect raw current reads as well as signal length, resulting in a shift in signal distributions (e.g., divergence between blue and orange). However, these signal shifts can be modest, as shown by the largely overlapping density plots for CACCC and CAm 5 CCC, making accurate prediction of modified bases a computational challenge. Plots were generated with Sequoia, an interactive visual analytics platform for interpretation and feature extraction from ONT sequencing datasets [ 138 ].

    Article Snippet: We aim to highlight the strengths and limitations of current methods regarding specificity, sensitivity, and reproducibility, with a particular focus on emerging single-molecule direct RNA sequencing by Nanopore.

    Techniques: Nanopore Sequencing, RNA Sequencing Assay, Sequencing, Generated, Modification, Chick Chorioallantoic Membrane Assay, Countercurrent Chromatography, Methylation Sequencing

    Oxford Nanopore Technology read length distribution and CHEF electrophoresis of DNA from mitochondrial enriched T. gondii cell fractions. (A) Profile of read counts plotted by length for mtDNA (top) and nuclear (bottom). Species and strains are as indicated in the legend. (B) Southern of a Contour-clamped homogeneous electric field gel electrophoresis of T. gondii total DNA (Lane 1) and mitochondrial-enriched DNA (Lane 2) probed with a 1012 bp section of the cob gene. DNA ladder is as indicated. A plot of the ME49 and Nc-1 nuclear reads is located at the bottom of Table S3 since they could not be plotted here due to difference in read count scale.

    Journal: bioRxiv

    Article Title: A novel fragmented mitochondrial genome in the protist pathogen Toxoplasma gondii and related tissue coccidia

    doi: 10.1101/2020.05.16.099366

    Figure Lengend Snippet: Oxford Nanopore Technology read length distribution and CHEF electrophoresis of DNA from mitochondrial enriched T. gondii cell fractions. (A) Profile of read counts plotted by length for mtDNA (top) and nuclear (bottom). Species and strains are as indicated in the legend. (B) Southern of a Contour-clamped homogeneous electric field gel electrophoresis of T. gondii total DNA (Lane 1) and mitochondrial-enriched DNA (Lane 2) probed with a 1012 bp section of the cob gene. DNA ladder is as indicated. A plot of the ME49 and Nc-1 nuclear reads is located at the bottom of Table S3 since they could not be plotted here due to difference in read count scale.

    Article Snippet: We used Nanopore sequencing precisely because each read is generated from an individual DNA molecule with the addition of any amplification steps.

    Techniques: Electrophoresis, Nucleic Acid Electrophoresis

    Nanopore and Illumina comparisons reveal sequence block order variability and decay with evolutionary time. (A-B) Two T. gondii ME49 Nanopore sequence reads were annotated with the gene sequences they contain. Reads and SBs are drawn to scale. The blocks are colored as shown in Table 1 . ‘Annotated genes’ track represents the annotation of the cytochrome genes and rRNA gene fragments on the Nanopore reads. The three ‘Paired-end Illumina DNA mapping’ tracks show paired-end read mapping of T. gondii Tg ME49 (SRR9200762), Tg RH (SRR521957) and N. caninum (Nc) LIV (ERR012900) mtDNA-specific reads. Mapping of Tg ME49 and Tg RH reads required 100% nucleotide identity whereas 1% mismatch was allowed for mapping Nc LIV reads. Reads were independently mapped to each of the Nanopore mtDNA reads and visualized using IGV. Red and blue lines below each read indicate the mapped Illumina paired-end reads.

    Journal: bioRxiv

    Article Title: A novel fragmented mitochondrial genome in the protist pathogen Toxoplasma gondii and related tissue coccidia

    doi: 10.1101/2020.05.16.099366

    Figure Lengend Snippet: Nanopore and Illumina comparisons reveal sequence block order variability and decay with evolutionary time. (A-B) Two T. gondii ME49 Nanopore sequence reads were annotated with the gene sequences they contain. Reads and SBs are drawn to scale. The blocks are colored as shown in Table 1 . ‘Annotated genes’ track represents the annotation of the cytochrome genes and rRNA gene fragments on the Nanopore reads. The three ‘Paired-end Illumina DNA mapping’ tracks show paired-end read mapping of T. gondii Tg ME49 (SRR9200762), Tg RH (SRR521957) and N. caninum (Nc) LIV (ERR012900) mtDNA-specific reads. Mapping of Tg ME49 and Tg RH reads required 100% nucleotide identity whereas 1% mismatch was allowed for mapping Nc LIV reads. Reads were independently mapped to each of the Nanopore mtDNA reads and visualized using IGV. Red and blue lines below each read indicate the mapped Illumina paired-end reads.

    Article Snippet: We used Nanopore sequencing precisely because each read is generated from an individual DNA molecule with the addition of any amplification steps.

    Techniques: Sequencing, Blocking Assay

    Annotated Toxoplasma gondii ME49 Nanopore mtDNA reads. Each panel represents the annotation of a single Nanopore read with SBs. There are no intervening nucleotides between SBs. The length of the read is indicated above the annotation. MtDNA-specific paired-end Illumina reads generated from T. gondii ME49 DNA (SRR9200762) were mapped to the Nanopore sequences and the alignment was visualized using the Integrated Genomics Viewer (IGV). Both Illumina read ends were required to map at 100% identity. A histogram of read abundance is shown in grey just below each annotated Nanopore read. Red and blue lines below each histogram indicate the mapped paired-end reads. Not all mapped reads are shown.

    Journal: bioRxiv

    Article Title: A novel fragmented mitochondrial genome in the protist pathogen Toxoplasma gondii and related tissue coccidia

    doi: 10.1101/2020.05.16.099366

    Figure Lengend Snippet: Annotated Toxoplasma gondii ME49 Nanopore mtDNA reads. Each panel represents the annotation of a single Nanopore read with SBs. There are no intervening nucleotides between SBs. The length of the read is indicated above the annotation. MtDNA-specific paired-end Illumina reads generated from T. gondii ME49 DNA (SRR9200762) were mapped to the Nanopore sequences and the alignment was visualized using the Integrated Genomics Viewer (IGV). Both Illumina read ends were required to map at 100% identity. A histogram of read abundance is shown in grey just below each annotated Nanopore read. Red and blue lines below each histogram indicate the mapped paired-end reads. Not all mapped reads are shown.

    Article Snippet: We used Nanopore sequencing precisely because each read is generated from an individual DNA molecule with the addition of any amplification steps.

    Techniques: Generated

    Full-length protein-encoding genes in the T. gondii mt genome are supported. Segments of a Nanopore read that can encode a full-length cytochrome gene are annotated with their sequence block name and shown below the schematic of each gene. Numbers below each gene schematic represent nucleotide start/stop positions of a sequence block on the gene. All blocks are in the forward orientation except block V in coxIII . All blocks shown participate fully in creation of the cytochrome sequence except block V where only a part of coxIII is used, and sequence blocks M and T where only a portion of the block contributes to encoding a cytochrome gene. MtDNA-specific paired-end Illumina DNA (SRR9200762) and RNA-seq (SRR6493545) reads were independently mapped to each of the cytochrome gene sequences and visualized using IGV. Red and blue lines below each gene indicate the mapped Illumina paired-ends. Both ends were required to map.

    Journal: bioRxiv

    Article Title: A novel fragmented mitochondrial genome in the protist pathogen Toxoplasma gondii and related tissue coccidia

    doi: 10.1101/2020.05.16.099366

    Figure Lengend Snippet: Full-length protein-encoding genes in the T. gondii mt genome are supported. Segments of a Nanopore read that can encode a full-length cytochrome gene are annotated with their sequence block name and shown below the schematic of each gene. Numbers below each gene schematic represent nucleotide start/stop positions of a sequence block on the gene. All blocks are in the forward orientation except block V in coxIII . All blocks shown participate fully in creation of the cytochrome sequence except block V where only a part of coxIII is used, and sequence blocks M and T where only a portion of the block contributes to encoding a cytochrome gene. MtDNA-specific paired-end Illumina DNA (SRR9200762) and RNA-seq (SRR6493545) reads were independently mapped to each of the cytochrome gene sequences and visualized using IGV. Red and blue lines below each gene indicate the mapped Illumina paired-ends. Both ends were required to map.

    Article Snippet: We used Nanopore sequencing precisely because each read is generated from an individual DNA molecule with the addition of any amplification steps.

    Techniques: Sequencing, Blocking Assay, RNA Sequencing Assay