Non-templated addition and template switching by Moloney murine leukemia virus (MMLV)-based reverse transcriptases co-occur and compete with each other

Wulf, Madalee G.; Maguire, Sean; Humbert, P.; Dai, Nan; Bei, Yanxia; Nichols, Nicole M.; Corrêa, Ivan R.; Guan, Shengxi

doi:10.1074/jbc.ra119.010676

Cited by 68 publications

(83 citation statements)

References 41 publications

(55 reference statements)

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“…The RAMPAGE protocol preserves the mRNA polarity in the resulting cDNA library by the directed introduction of Illumina-sequencing adapters for Read 1 at the 5′ end via a template switching oligo (TSO), and for Read 2 toward the 3′ end of the mRNA transcript via a randomly-priming reverse-transcription (RT) primer ( Figure 1A , panel 3 ). RAMPAGE employs two approaches to enrich for 5′-intact mRNA based on the presence of the 5′-m7G cap structure (cap): by (1) introducing the 5′-sequencing adapter via template switching, which preferentially occurs at the cap ( Wulf et al., 2019 ), and by (2) biotinylation of the cap, followed by capture on streptavidin-coated beads ( Figure 1A , panels 3–5 ). Upon library amplification and paired-end sequencing, reads from both replicates of all three samples were mapped to the ME49 genome assembly (ToxoDB, version 45), and reads originating from ribosomal RNA (rRNA) contamination were removed ( Figure 1A , panels 6–8; Figure 1B ).…”

Section: Resultsmentioning

confidence: 99%

High-Resolution Mapping of Transcription Initiation in the Asexual Stages of Toxoplasma gondii

Markus

Waldman

Lorenzi

et al. 2021

Front. Cell. Infect. Microbiol.

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Toxoplasma gondii is a common parasite of humans and animals, causing life-threatening disease in the immunocompromized, fetal abnormalities when contracted during gestation, and recurrent ocular lesions in some patients. Central to the prevalence and pathogenicity of this protozoan is its ability to adapt to a broad range of environments, and to differentiate between acute and chronic stages. These processes are underpinned by a major rewiring of gene expression, yet the mechanisms that regulate transcription in this parasite are only partially characterized. Deciphering these mechanisms requires a precise and comprehensive map of transcription start sites (TSSs); however, Toxoplasma TSSs have remained incompletely defined. To address this challenge, we used 5′-end RNA sequencing to genomically assess transcription initiation in both acute and chronic stages of Toxoplasma. Here, we report an in-depth analysis of transcription initiation at promoters, and provide empirically-defined TSSs for 7603 (91%) protein-coding genes, of which only 1840 concur with existing gene models. Comparing data from acute and chronic stages, we identified instances of stage-specific alternative TSSs that putatively generate mRNA isoforms with distinct 5′ termini. Analysis of the nucleotide content and nucleosome occupancy around TSSs allowed us to examine the determinants of TSS choice, and outline features of Toxoplasma promoter architecture. We also found pervasive divergent transcription at Toxoplasma promoters, clustered within the nucleosomes of highly-symmetrical phased arrays, underscoring chromatin contributions to transcription initiation. Corroborating previous observations, we asserted that Toxoplasma 5′ leaders are among the longest of any eukaryote studied thus far, displaying a median length of approximately 800 nucleotides. Further highlighting the utility of a precise TSS map, we pinpointed motifs associated with transcription initiation, including the binding sites of the master regulator of chronic-stage differentiation, BFD1, and a novel motif with a similar positional arrangement present at 44% of Toxoplasma promoters. This work provides a critical resource for functional genomics in Toxoplasma, and lays down a foundation to study the interactions between genomic sequences and the regulatory factors that control transcription in this parasite.

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Section: Resultsmentioning

confidence: 99%

High-Resolution Mapping of Transcription Initiation in the Asexual Stages of Toxoplasma gondii

Markus

Waldman

Lorenzi

et al. 2021

Front. Cell. Infect. Microbiol.

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“…We generated a comprehensive full-length transcript database in B. napus by pooling RNA from five tissues with four cDNA libraries with sizes ranging from < 1 kb to > 3 kb using the PacBio Iso-Seq approach. Recently, it has been reported that template switching take place in a sequential process, allowing the reverse transcriptase (RT) to switch templates and continue copying the templateswitching oligo sequence (Wulf et al, 2019). Moreover, the presence of a 5 0 cap can enhance template switching efficiency, allowing cDNAs from uncapped RNA to also be tagged with a 5 0 adapter sequence.…”

Section: Discussion and Future Directionsmentioning

confidence: 99%

A global survey of the transcriptome of allopolyploid Brassica napus based on single‐molecule long‐read isoform sequencing and Illumina‐based RNA sequencing data

et al. 2020

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SUMMARY Brassica napus is a recent allopolyploid derived from the hybridization of Brassica rapa (ArAr) and Brassica oleracea (CoCo). Because of the high sequence similarity between the An and Cn subgenomes, it is difficult to provide an accurate landscape of the whole transcriptome of B. napus. To overcome this problem, we applied a single‐molecule long‐read isoform sequencing (Iso‐Seq) technique that can produce long reads to explore the complex transcriptome of B. napus at the isoform level. From the Iso‐Seq data, we obtained 147 698 non‐redundant isoforms, capturing 37 403 annotated genes. A total of 18.1% (14 934/82 367) of the multi‐exonic genes showed alternative splicing (AS). In addition, we identified 549 long non‐coding RNAs, the majority of which displayed tissue‐specific expression profiles, and detected 7742 annotated genes that possessed isoforms containing alternative polyadenylation sites. Moreover, 31 591 AS events located in open reading frames (ORFs) lead to potential protein isoforms by in‐frame or frameshift changes in the ORF. Illumina RNA sequencing of five tissues that were pooled for Iso‐Seq was also performed and showed that 69% of the AS events were tissue‐specific. Our data provide abundant transcriptome resources for a transcript isoform catalog of B. napus, which will facilitate genome reannotation, strengthen our understanding of the B. napus transcriptome and be applied for further functional genomic research.

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“…The SMARTer small RNA kit (Takara) uses poly-adenylation followed by reverse transcription and template switching to make ligation-free libraries. While this technique does eliminate ligation associated biases, template switching on uncapped sRNAs itself has some sequence bias ( 17 ). Furthermore, for reasons that are not well understood, template switching has a very low detection sensitivity for miRNAs compared to ligation-based approaches when performed on total RNA, due to a large amplification of background RNA such as rRNA ( 18–20 ).…”

Section: Introductionmentioning

confidence: 99%

A low-bias and sensitive small RNA library preparation method using randomized splint ligation

Maguire

Lohman

Guan

2020

Nucleic Acids Research

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Small RNAs are important regulators of gene expression and are involved in human development and disease. Next generation sequencing (NGS) allows for scalable, genome-wide studies of small RNA; however, current methods are challenged by low sensitivity and high bias, limiting their ability to capture an accurate representation of the cellular small RNA population. Several studies have shown that this bias primarily arises during the ligation of single-strand adapters during library preparation, and that this ligation bias is magnified by 2′-O-methyl modifications (2′OMe) on the 3′ terminal nucleotide. In this study, we developed a novel library preparation process using randomized splint ligation with a cleavable adapter, a design which resolves previous challenges associated with this ligation strategy. We show that a randomized splint ligation based workflow can reduce bias and increase the sensitivity of small RNA sequencing for a wide variety of small RNAs, including microRNA (miRNA) and tRNA fragments as well as 2′OMe modified RNA, including Piwi-interacting RNA and plant miRNA. Finally, we demonstrate that this workflow detects more differentially expressed miRNA between tumorous and matched normal tissues. Overall, this library preparation process allows for highly accurate small RNA sequencing and will enable studies of 2′OMe modified RNA with new levels of detail.

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Non-templated addition and template switching by Moloney murine leukemia virus (MMLV)-based reverse transcriptases co-occur and compete with each other

Cited by 68 publications

References 41 publications

High-Resolution Mapping of Transcription Initiation in the Asexual Stages of Toxoplasma gondii

High-Resolution Mapping of Transcription Initiation in the Asexual Stages of Toxoplasma gondii

A global survey of the transcriptome of allopolyploid Brassica napus based on single‐molecule long‐read isoform sequencing and Illumina‐based RNA sequencing data

A low-bias and sensitive small RNA library preparation method using randomized splint ligation

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