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Ncec

ABSTRACT: Enabling high-accuracy long-read amplicon sequences using unique molecular identifiers with Nanopore or PacBio sequencing

PROVIDER: PRJEB32674 | ENA |

REPOSITORIES: ENA

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			Action	DRS
		Other
		Other
	smkj407_zymo_D6306_rrna_umi_pb_ccs_var.fasta.gz	Fasta.gz
	smkj407_zymo_D6306_rrna_umi_pb_var.fasta.gz	Fasta.gz
	smkj412_zymo_D6306_rrna_umi_ont_r10_g344hac_var.fasta.gz	Fasta.gz

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longread_umi

Project description:Enabling high-accuracy long-read amplicon sequences using unique molecular identifiers with Nanopore or PacBio sequencing

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High throughput error correction using dual nucleotide dimer blocks allows direct single-cell nanopore transcriptome sequencing

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Anchor-Enhanced Bead Design for Reduced Oligonucleotide Synthesis Errors in Single-cell sequencing

Project description:Single-cell transcriptomics, reliant on the incorporation of barcodes and unique molecular identifiers (UMIs) into captured polyA+ mRNA, faces a significant challenge due to synthesis errors in oligonucleotide capture sequences. These inaccuracies, which are especially problematic in long-read sequencing, impair the precise identification of sequences and result in inaccuracies in UMI deduplication. To mitigate this issue, we have modified the oligonucleotide capture design, which integrates an interposed anchor between the barcode and UMI, and a 'V' base anchor adjacent to the polyA capture region. This configuration is devised to ensure compatibility with both short and long-read sequencing technologies, facilitating improved UMI recovery and enhanced feature detection, thereby improving the efficacy of droplet-based sequencing methods.

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Reference Short-Read Transcriptomes of Human Peripheral Blood Lymphocytes (Illumina RNA-Seq, Matched to PacBio Iso-Seq Data)

Project description:Long-read sequencing technologies such as Iso-Seq (PacBio Inc.) generate highly accurate sequences of full-length mRNA transcript isoforms. Long-read transcriptomics may be especially useful in the context of lymphocyte functional plasticity as it relates to human health and disease. However, no long-read isoform-aware reference transcriptomes of human circulating lymphocytes seem to be publicly available despite being valuable as benchmarks in a variety of transcriptomic studies. To begin to fill this gap, we purified four lymphocyte subsets (CD4 T, CD8 T, NK, and Pan B cells) from the peripheral blood of a healthy male donor and obtained high-quality RNA (RIN>8) for PacBio Iso-Seq analysis and parallel RNA-Seq analysis.

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A technology-agnostic long-read analysis pipeline for transcriptome discovery and quantification

Project description:Alternative splicing is widely acknowledged to be a crucial regulator of gene expression and is a key contributor to both normal developmental processes and disease states. While cost-effective and accurate for quantification, short-read RNA-seq lacks the ability to resolve full-length transcript isoforms despite increasingly sophisticated computational methods. Long-read sequencing platforms such as Pacific Biosciences (PacBio) and Oxford Nanopore (ONT) bypass the transcript reconstruction challenges of short-reads. Here we describe TALON, the ENCODE4 pipeline for analyzing PacBio cDNA and ONT direct-RNA transcriptomes. We apply TALON to three human ENCODE Tier 1 cell lines and show that while both technologies perform well at full-transcript discovery and quantification, each one displayed distinct artifacts. We further apply TALON to mouse cortical and hippocampal transcriptomes and find that a substantial proportion of neuronal genes have more reads associated with novel isoforms than with annotated ones. These data show that TALON is a technology-agnostic long-read transcriptome discovery and quantification pipeline capable of tracking both known and novel transcript models, as well as their expression levels, across datasets for both simple studies and in larger projects. These properties will enable TALON users to move beyond the limitations of short-read data to perform isoform discovery and quantification in a uniform manner on existing and future long-read platforms.

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