OVERCOMING FALSE POSITIVES OF REVERSE TRANSCRIPTION AT THE DETECTION OF CHIMERIC RNAS

2021 ◽  
Author(s):  
Александр Андреевич Гордеев ◽  
Елена Владимировна Четверина ◽  
Марина Витальевна Фалалеева ◽  
Александр Борисович Четверин
Keyword(s):  
Reverse Transcriptase ◽  
Reverse Transcription ◽  
False Positives ◽  
Chimeric Rnas ◽  
Reverse Transcription Reaction ◽  
Template Switch

Работа посвящена исследованию формирования ложных химерных кДНК в результате смены матриц обратной транскриптазой. Показано, что, изменяя ряд параметров реакции обратной транскрипции, можно существенно уменьшить частоту ложноположительных результатов при выявлении истинных химерных РНК. Полученные результаты позволяют улучшить качество анализа транскриптомов и диагностики заболеваний, ассоциированных с образованием химерных РНК. This work is aimed at the study of formation of false chimeric cDNA as a result of template switch by reverse transcriptase. It is shown that by manipulating a number of parameters of the reverse transcription reaction, it is possible to significantly reduce the frequency of false-positives in the detection of true chimeric RNAs. The results allow to improve the quality of the analysis of transcriptomes and of the diagnostics of diseases associated with the formation of chimeric RNAs.

scholarly journals Identification and removal of sequencing artifacts produced by mispriming during reverse transcription in multiple RNA-seq technologies

2018 ◽  
Author(s):  
Haridha Shivram ◽  
Vishwanath R. Iyer
Keyword(s):  
Reverse Transcriptase ◽  
Rna Sequencing ◽  
Reverse Transcription ◽  
Group Ii Intron ◽  
Computational Pipeline ◽  
Rna Seq ◽  
Sequencing Data ◽  
Analysis Pipeline ◽  
Specific Priming

AbstractThe quality of RNA sequencing data relies on specific priming by the primer used for reverse transcription (RT-primer). Non-specific annealing of the RT-primer to the RNA template can generate reads with incorrect cDNA ends and can cause misinterpretation of data (RT mispriming). This kind of artifact in RNA-seq based technologies is underappreciated and currently no adequate tools exist to computationally remove them from published datasets. We show that mispriming can occur with as little as 2 bases of complementarity at the 3’ end of the primer followed by intermittent regions of complementarity. We also provide a computational pipeline that identifies cDNA reads produced from RT mispriming, allowing users to filter them out from any aligned dataset. Using this analysis pipeline, we identify thousands of mispriming events in a dozen published datasets from diverse technologies including short RNA-seq, total/mRNA-seq, HITS-CLIP and GRO-seq. We further show how RT-mispriming can lead to misinterpretation of data. In addition to providing a solution to computationally remove RT-misprimed reads, we also propose an experimental solution to avoid RT-mispriming by performing RNA-seq using thermostable group II intron derived reverse transcriptase (TGIRT-seq).

Optimisation of reverse transcription can improve the sensitivity of RT-PCR for detection of classical swine fever virus

2010 ◽  
Vol 58 (2) ◽  
pp. 257-264 ◽  
Author(s):  
Katarzyna Podgórska ◽  
Tomasz Stadejek
Keyword(s):  
Reverse Transcriptase ◽  
Reverse Transcription ◽  
Classical Swine Fever Virus ◽  
Animal Health ◽  
Thermal Conditions ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Diagnostic Methods ◽  
Rt Pcr ◽  
Reverse Transcription Reaction

Classical swine fever is a highly contagious, notifiable disease of pigs and wild boars listed by the World Organisation for Animal Health (OIE). Therefore, methods employed in the diagnosis of CSF should be fast, sensitive and specific. The aim of this study was optimisation of the reverse transcription reaction to increase the sensitivity of real-time RT-PCR for the detection of classical swine fever virus, the aetiological agent of the disease. The efficiency of reverse transcription reaction was compared including a range of reverse transcriptases, thermal conditions and priming methods based on results obtained in the following realtime PCR. Depending on catalysis and the priming method used in the study a significant diversity of results was observed. The best efficacy of reverse transcription was obtained using SuperScript II reverse transcriptase and priming with random nonamers and reverse, gene-specific primer. This combination improved the sensitivity of RT-PCR nearly 1000 times as compared to the method with AMV reverse transcriptase coupled with random hexamers. In summary, this study has demonstrated that the optimisation of reverse transcription can contribute to a higher sensitivity of RT-PCR diagnostic methods.

scholarly journals Intrinsic properties of reverse transcriptase in reverse transcription

1989 ◽  
Vol 264 (31) ◽  
pp. 18808-18817 ◽  
Author(s):  
F Oyama ◽  
R Kikuchi ◽  
R J Crouch ◽  
T Uchida
Keyword(s):  
Reverse Transcriptase ◽  
Reverse Transcription ◽  
Intrinsic Properties

scholarly journals High-resolution view of HIV-1 reverse transcriptase initiation complexes and inhibition by NNRTI drugs

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Betty Ha ◽  
Kevin P. Larsen ◽  
Jingji Zhang ◽  
Ziao Fu ◽  
Elizabeth Montabana ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Viral Entry ◽  
Reverse Transcription ◽  
Molecular Mechanisms ◽  
Dissociation Kinetics ◽  
Structural Mechanism ◽  
Medium Resolution ◽  
Kinetics Of ◽  
Hiv 1

AbstractReverse transcription of the HIV-1 viral RNA genome (vRNA) is an integral step in virus replication. Upon viral entry, HIV-1 reverse transcriptase (RT) initiates from a host tRNALys3 primer bound to the vRNA genome and is the target of key antivirals, such as non-nucleoside reverse transcriptase inhibitors (NNRTIs). Initiation proceeds slowly with discrete pausing events along the vRNA template. Despite prior medium-resolution structural characterization of reverse transcriptase initiation complexes (RTICs), higher-resolution structures of the RTIC are needed to understand the molecular mechanisms that underlie initiation. Here we report cryo-EM structures of the core RTIC, RTIC–nevirapine, and RTIC–efavirenz complexes at 2.8, 3.1, and 2.9 Å, respectively. In combination with biochemical studies, these data suggest a basis for rapid dissociation kinetics of RT from the vRNA–tRNALys3 initiation complex and reveal a specific structural mechanism of nucleic acid conformational stabilization during initiation. Finally, our results show that NNRTIs inhibit the RTIC and exacerbate discrete pausing during early reverse transcription.

The Mauriceville plasmid of Neurospora crassa: characterization of a novel reverse transcriptase that begins cDNA synthesis at the 3' end of template RNA

1992 ◽  
Vol 12 (11) ◽  
pp. 5131-5144
Author(s):  
H Wang ◽  
J C Kennell ◽  
M T Kuiper ◽  
J R Sabourin ◽  
R Saldanha ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Reverse Transcription ◽  
Full Length ◽  
Minus Strand ◽  
Ribonucleoprotein Particle ◽  
Cdna Synthesis ◽  
Reverse Transcriptases ◽  
In Vitro Transcripts

The Mauriceville and Varkud plasmids are retroid elements that propagate in the mitochondria of some Neurospora spp. strains. Previous studies of endogenous reactions in ribonucleoprotein particle preparations suggested that the plasmids use a novel mechanism of reverse transcription that involves synthesis of a full-length minus-strand DNA beginning at the 3' end of the plasmid transcript, which has a 3' tRNA-like structure (M. T. R. Kuiper and A. M. Lambowitz, Cell 55:693-704, 1988). In this study, we developed procedures for releasing the Mauriceville plasmid reverse transcriptase from mitochondrial ribonucleoprotein particles and partially purifying it by heparin-Sepharose chromatography. By using these soluble preparations, we show directly that the Mauriceville plasmid reverse transcriptase synthesizes full-length cDNA copies of in vitro transcripts beginning at the 3' end and has a preference for transcripts having the 3' tRNA-like structure. Further, unlike retroviral reverse transcriptases, the Mauriceville plasmid reverse transcriptase begins cDNA synthesis directly opposite the 3'-terminal nucleotide of the template RNA. The ability to initiate cDNA synthesis directly at the 3' end of template RNAs may also be relevant to the mechanisms of reverse transcription used by LINEs, group II introns, and other non-long terminal repeat retroid elements.

scholarly journals A Novel Leu92 Mutant of HIV-1 Reverse Transcriptase with a Selective Deficiency in Strand Transfer Causes a Loss of Viral Replication

2015 ◽  
Vol 89 (16) ◽  
pp. 8119-8129 ◽  
Author(s):  
Eytan Herzig ◽  
Nickolay Voronin ◽  
Nataly Kucherenko ◽  
Amnon Hizi
Keyword(s):  
Life Cycle ◽  
Viral Replication ◽  
Reverse Transcriptase ◽  
Dna Polymerase ◽  
Reverse Transcription ◽  
Polymerase Activity ◽  
Rnase H ◽  
Strand Transfer ◽  
Hiv 1

ABSTRACTThe process of reverse transcription (RTN) in retroviruses is essential to the viral life cycle. This key process is catalyzed exclusively by the viral reverse transcriptase (RT) that copies the viral RNA into DNA by its DNA polymerase activity, while concomitantly removing the original RNA template by its RNase H activity. During RTN, the combination between DNA synthesis and RNA hydrolysis leads to strand transfers (or template switches) that are critical for the completion of RTN. The balance between these RT-driven activities was considered to be the sole reason for strand transfers. Nevertheless, we show here that a specific mutation in HIV-1 RT (L92P) that does not affect the DNA polymerase and RNase H activities abolishes strand transfer. There is also a good correlation between this complete loss of the RT's strand transfer to the loss of the DNA clamp activity of the RT, discovered recently by us. This finding indicates a mechanistic linkage between these two functions and that they are both direct and unique functions of the RT (apart from DNA synthesis and RNA degradation). Furthermore, when the RT's L92P mutant was introduced into an infectious HIV-1 clone, it lost viral replication, due to inefficient intracellular strand transfers during RTN, thus supporting thein vitrodata. As far as we know, this is the first report on RT mutants that specifically and directly impair RT-associated strand transfers. Therefore, targeting residue Leu92 may be helpful in selectively blocking this RT activity and consequently HIV-1 infectivity and pathogenesis.IMPORTANCEReverse transcription in retroviruses is essential for the viral life cycle. This multistep process is catalyzed by viral reverse transcriptase, which copies the viral RNA into DNA by its DNA polymerase activity (while concomitantly removing the RNA template by its RNase H activity). The combination and balance between synthesis and hydrolysis lead to strand transfers that are critical for reverse transcription completion. We show here for the first time that a single mutation in HIV-1 reverse transcriptase (L92P) selectively abolishes strand transfers without affecting the enzyme's DNA polymerase and RNase H functions. When this mutation was introduced into an infectious HIV-1 clone, viral replication was lost due to an impaired intracellular strand transfer, thus supporting thein vitrodata. Therefore, finding novel drugs that target HIV-1 reverse transcriptase Leu92 may be beneficial for developing new potent and selective inhibitors of retroviral reverse transcription that will obstruct HIV-1 infectivity.

scholarly journals Determination of Bovine Rotavirus G and P Serotypes in Italy by PCR

1999 ◽  
Vol 37 (12) ◽  
pp. 3879-3882 ◽  
Author(s):  
E. Falcone ◽  
M. Tarantino ◽  
L. Di Trani ◽  
P. Cordioli ◽  
A. Lavazza ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Clinical Signs ◽  
Specific Primers ◽  
Bovine Rotavirus ◽  
Reverse Transcription Pcr ◽  
Rotavirus Vaccines ◽  
Intestinal Contents ◽  
Group A

Determination of the G and P serotypes of group A bovine rotaviruses from 149 samples of feces or intestinal contents collected from calves showing clinical signs of neonatal diarrhea was performed by a nested reverse transcription-PCR typing assay. The G6 serotype was the most prevalent, accounting for viruses in 55.7% of the samples; viruses of the G10 and G8 serotypes were found in 34.9 and 4.7% of the samples, respectively. The virus in one sample (0.7%) was not classified due to concomitant infection with G6 and G8 strains, whereas viruses in six samples (4.0%) could not be characterized with any of the three G serotype-specific primers selected for the present study. When examined for their P-serotype specificities, viruses in 55 and 42.3% of the samples were characterized as P[11] and P[5], respectively, no P[1] serotype was identified, and viruses in 2.7% of the samples could not be classified due to multiple reactivity with both P[5]- and P[11]-specific primers. Various combinations of G and P serotypes were observed, the most frequent being G6,P[5] (38.3%), G10,P[11] (31.5%), and G6,P[11] (15.4%). The results of the present study, while contributing to a better understanding of the epidemiology of bovine rotaviruses in Italy, address the relevance of serotype specificity with regard to the constancy of the quality of bovine rotavirus vaccines under different field conditions.

scholarly journals Binding and kinetic properties of HIV-1 reverse transcriptase markedly differ during initiation and elongation of reverse transcription.

1996 ◽  
Vol 15 (24) ◽  
pp. 7178-7187 ◽  
Author(s):  
J. M. Lanchy ◽  
C. Ehresmann ◽  
S. F. Le Grice ◽  
B. Ehresmann ◽  
R. Marquet
Keyword(s):  
Reverse Transcriptase ◽  
Reverse Transcription ◽  
Kinetic Properties ◽  
Hiv 1
Sign in / Sign up

Export Citation Format

Share Document