Polyadenylation at the 3 ′ ′ ′ ′ ′ -end is a major regulator of messenger RNA and its length is known to affect nuclear export, stability, and translation, among others. Only recently have strategies emerged that allow for genome-wide poly(A) length assessment. These methods identify genes connected to poly(A) tail measurements indirectly by short-read alignment to genetic 3 ′ ′ ′ ′ ′ -ends. Concurrently, Oxford Nanopore Technologies (ONT) established full-length isoform-specific RNA sequencing containing the entire poly(A) tail. However, assessing poly(A) length through base-calling has so far not been possible due to the inability to resolve long homopolymeric stretches in ONT sequencing. Here we present tailfindr, an R package to estimate poly(A) tail length on ONT long-read sequencing data. tailfindr operates on unaligned, base-called data. It measures poly(A) tail length from both native RNA and DNA sequencing, which makes poly(A) tail studies by full-length cDNA approaches possible for the first time. We assess tailfindr's performance across different poly(A) lengths, demonstrating that tailfindr is a versatile tool providing poly(A) tail estimates across a wide range of sequencing conditions.
RNA polyadenylation plays a central role in RNA maturation, fate, and stability. In response to developmental cues, polyA tail lengths can vary, affecting the translation efficiency and stability of mRNAs. Here we develop Nanopore 3′ end-capture sequencing (Nano3P-seq), a method that relies on nanopore cDNA sequencing to simultaneously quantify RNA abundance, tail composition, and tail length dynamics at per-read resolution. By employing a template-switching-based sequencing protocol, Nano3P-seq can sequence RNA molecule from its 3′ end, regardless of its polyadenylation status, without the need for PCR amplification or ligation of RNA adapters. We demonstrate that Nano3P-seq provides quantitative estimates of RNA abundance and tail lengths, and captures a wide diversity of RNA biotypes. We find that, in addition to mRNA and long non-coding RNA, polyA tails can be identified in 16S mitochondrial ribosomal RNA in both mouse and zebrafish models. Moreover, we show that mRNA tail lengths are dynamically regulated during vertebrate embryogenesis at an isoform-specific level, correlating with mRNA decay. Finally, we demonstrate the ability of Nano3P-seq in capturing non-A bases within polyA tails of various lengths, and reveal their distribution during vertebrate embryogenesis. Overall, Nano3P-seq is a simple and robust method for accurately estimating transcript levels, tail lengths, and tail composition heterogeneity in individual reads, with minimal library preparation biases, both in the coding and non-coding transcriptome.
Visual attention is used to selectively filter relevant information depending on current task demands and goals. Visual attention is called object-based attention when it is directed to coherent forms or objects in the visual field. This study used real-time functional magnetic resonance imaging for moment-to-moment decoding of attention to spatially overlapped objects belonging to two different object categories. First, a whole-brain classifier was trained on pictures of faces and places. Subjects then saw transparently overlapped pictures of a face and a place, and attended to only one of them while ignoring the other. The category of the attended object, face or place, was decoded on a scan-by-scan basis using the previously trained decoder. The decoder performed at 77.6% accuracy indicating that despite competing bottom-up sensory input, object-based visual attention biased neural patterns towards that of the attended object. Furthermore, a comparison between different classification approaches indicated that the representation of faces and places is distributed rather than focal. This implies that real-time decoding of object-based attention requires a multivariate decoding approach that can detect these distributed patterns of cortical activity.
Polyadenylation at the 3’-end is a major regulator of messenger RNA and its length is known to affect nuclear export, stability and translation, among others. Only recently, strategies have emerged that allow for genome-wide poly(A) length assessment. These methods identify genes connected to poly(A) tail measurements indirectly by short-read alignment to genetic 3’-ends. Concurrently Oxford Nanopore Technologies (ONT) established full-length isoform RNA sequencing containing the entire poly(A) tail. However, assessing poly(A) length through basecalling has so far not been possible due the inability to resolve long homopolymeric stretches in ONT sequencing.Here we presenttailfindr, an R package to estimate poly(A) tail length on ONT long-read sequencing data.tailfindroperates on unaligned, basecalled data. It measures poly(A) tail length from both native RNA and DNA sequencing, which makes poly(A) tail studies by full-length cDNA approaches possible for the first time. We assesstailfindr’sperformance across different poly(A) lengths, demonstrating thattailfindris a versatile tool providing poly(A) tail estimates across a wide range of sequencing conditions.
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