11 research outputs found
Systematic assessment of long-read RNA-seq methods for transcript identification and quantification
The Long-read RNA-Seq Genome Annotation Assessment Project (LRGASP) Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. The consortium generated over 427 million long-read sequences from cDNA and direct RNA datasets, encompassing human, mouse, and manatee species, using different protocols and sequencing platforms. These data were utilized by developers to address challenges in transcript isoform detection and quantification, as well as de novo transcript isoform identification. The study revealed that libraries with longer, more accurate sequences produce more accurate transcripts than those with increased read depth, whereas greater read depth improved quantification accuracy. In well-annotated genomes, tools based on reference sequences demonstrated the best performance. When aiming to detect rare and novel transcripts or when using reference-free approaches, incorporating additional orthogonal data and replicate samples are advised. This collaborative study offers a benchmark for current practices and provides direction for future method development in transcriptome analysis
Application of Long-Read Sequencing to Modified Nucleotides for Detecting Chromatin Accessibility
Application of Long-Read Sequencing to Modified Nucleotides for Detecting Chromatin Accessibility
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Application of Long-Read Sequencing to Modified Nucleotides for Detecting Chromatin Accessibility
Nucleosomes provide an additional layer of gene regulation by regulating chromatin ac-cessibility. We have found that factors involved in regulating nucleosome positioning,
such as SMARCA4, are recurrently mutated in lung adenocarcinoma and can correlate
with alternative splicing changes. As a result, it is crucial to understand nucleosome po-
sitioning across an entire gene body to understand how they interact to cause changes
in splicing. The key to being able to understand this is using long-read sequencing
methods to understand the positioning of nucleosomes at once. My thesis focuses on
developing methods and computational tools to understand nucleosome positioning with
long reads. I developed a sequencing approach called Add-seq that uses a small molecule
called angelicin to label accessible regions and determine those positions using nanopore
sequencing. Based on the analyses for Add-seq, I also developed a toolkit called cawlr.
This computational pipeline can automate calling nucleosomes on single molecules from
any long-read sequencing technology. This work provides new ways of looking at nucle-
osomes and understanding their positioning in the context of other biological processes
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Probing chromatin accessibility with small molecule DNA intercalation and nanopore sequencing
Genome-wide identification of chromatin organization and structure has been generally probed by measuring accessibility of the underlying DNA to nucleases or methyltransferases. These methods either only observe the positioning of a single nucleosome or rely on large enzymes to modify or cleave the DNA. We developed adduct sequencing (Add-seq), a method to probe chromatin accessibility by treating chromatin with the small molecule angelicin, which preferentially intercalates into DNA not bound to core nucleosomes. We show that Nanopore sequencing of the angelicin-modified DNA is possible and allows visualization and analysis of long single molecules with distinct chromatin structure. The angelicin modification can be detected from the Nanopore current signal data using a neural network model trained on unmodified and modified chromatin-free DNA. Applying Add-seq to Saccharomyces cerevisiae nuclei, we identified expected patterns of accessibility around annotated gene loci in yeast. We also identify individual clusters of single molecule reads displaying different chromatin structure at specific yeast loci, which demonstrates heterogeneity in the chromatin structure of the yeast population. Thus, using Add-seq, we are able to profile DNA accessibility in the yeast genome across long molecules