Biology subjects

Niazi, A. M.

Publications and source records attributed to Niazi, A. M..

3 recordsLinked to original sources

bioRxiv2024

Nano3P-seq: charting the coding and non-coding transcriptome at single molecule resolution

RNA polyadenylation is crucial for RNA maturation, stability and function, with polyA tail lengths significantly influencing mRNA translation, efficiency and decay. Here, we provide a step-by-step protocol to perform Nanopore 3 end-capture sequencing (Nano3P-seq), a nanopore-based cDNA sequencing method to simultaneously capture RNA abundances, tail composition and tail length estimates at single-molecule resolution. Taking advantage of a template switching-based protocol, Nano3P-seq can sequence any RNA molecule from its 3 end, regardless of its polyadenylation status, without the need for PCR amplification or RNA adapter ligation. We provide an updated Nano3P-seq protocol that is compatible with R10.4 flowcells, as well as compatible software for polyA tail length and content prediction, which we term PolyTailor. We demonstrate that PolyTailor provides accurate estimates of transcript abundances, tail lengths and content information, while capturing both coding and non-coding RNA biotypes, including mRNAs, snRNAs, and rRNAs. This method can be applied to any RNA sample of interest (e.g. poly(A)-selected, ribodepleted, total RNA), and can be completed in one day. The Nano3P-seq protocol can be performed by researchers with moderate experience in molecular biology techniques and nanopore sequencing library preparation, and basic knowledge of linux bash syntax and R programming. This protocol makes Nano3P-seq accessible and easy to implement by future users aiming to study the tail dynamics and heterogeneity of both coding and non-coding transcriptome in a comprehensive and reproducible manner. Key PapersBe[g]ik O, Diensthuber G, Liu H, Delgado-Tejedor A, Kontur C, Niazi AM, Valen E, Giraldez AJ, Beaudoin JD, Mattick JS, Novoa EM. Nano3P-seq: transcriptome-wide analysis of gene expression and tail dynamics using end-capture nanopore cDNA sequencing. Nature Methods 20, 75-85 (2023). https://doi.org/10.1038/s41592-022-01714-w Delgado-Tejedor A, Medina M, Begik O, Cozzuto L, Lopez J, Blanco B, Ponomarenko J, Novoa EM. Native RNA nanopore sequencing reveals antibiotic-induced loss of rRNA modifications in the A- and P-sites. NatComm 15, 10054 (2024). https://doi.org/10.1038/s41467-024-54368-x

molecular biology↗

bioRxiv2023

Generation of a porcine cell line stably expressing pig TMPRSS2 for efficient isolation of viruses from pigs with respiratory diseases

Pigs are important animals for meat production but can carry several zoonotic diseases, including Japanese encephalitis virus, Nipah virus, and influenza viruses. Several Orthomyxoviridae and Coronavirinae respiratory viruses require cleavage of envelope proteins to acquire viral infectivity and consequently need a host protease or the addition of exogenous trypsin for efficient propagation. Host TMPRSS2 is a key protease responsible for viral cleavage. Stable expression of human TMPRSS2 in African green monkey-derived Vero cells can enhance the porcine epidemic diarrhea virus. However, considering the narrow host tropism of viruses, a porcine cell line expressing pig TMPRSS2 could be optimal for replicating pig-derived viruses. Herein, we generated and evaluated a pig-derived PK-15 cell line stably expressing pig TMPRSS2. This cell line markedly and specifically enhanced the growth of influenza viruses. Therefore, PK-15 cells expressing pig TMPRSS2 could be a valuable and promising tool for virus isolation, vaccine production, and virological studies.

microbiology↗

bioRxiv2019

tailfindr: Alignment-free poly(A) length measurement for Oxford Nanopore RNA and DNA sequencing

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.\n\nHere we present tailfindr, an R package to estimate poly(A) tail length on ONT long-read sequencing data. tailfindr operates 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 assess tailfindrs 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.

bioinformatics↗