adapter ligation
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2022 ◽  
Vol 0 (0) ◽  
Author(s):  
V. Janett Olzog ◽  
Lena I. Freist ◽  
Robin Goldmann ◽  
Jörg Fallmann ◽  
Christina E. Weinberg

Abstract Self-cleaving ribozymes are catalytic RNAs and can be found in all domains of life. They catalyze a site-specific cleavage that results in a 5′ fragment with a 2′,3′ cyclic phosphate (2′,3′ cP) and a 3′ fragment with a 5′ hydroxyl (5′ OH) end. Recently, several strategies to enrich self-cleaving ribozymes by targeted biochemical methods have been introduced by us and others. Here, we develop an alternative strategy in which 5ʹ OH RNAs are specifically ligated by RtcB ligase, which first guanylates the 3′ phosphate of the adapter and then ligates it directly to RNAs with 5′ OH ends. Our results demonstrate that adapter ligation to highly structured ribozyme fragments is much more efficient using the thermostable RtcB ligase from Pyrococcus horikoshii than the broadly applied Escherichia coli enzyme. Moreover, we investigated DNA, RNA and modified RNA adapters for their suitability in RtcB ligation reactions. We used the optimized RtcB-mediated ligation to produce RNA-seq libraries and captured a spiked 3ʹ twister ribozyme fragment from E. coli total RNA. This RNA-seq-based method is applicable to detect ribozyme fragments as well as other cellular RNAs with 5ʹ OH termini from total RNA.


2021 ◽  
Vol 17 (12) ◽  
pp. e1009632
Author(s):  
Thomas W. Christy ◽  
Catherine A. Giannetti ◽  
Alain Laederach ◽  
Kevin M. Weeks

SHAPE-JuMP is a concise strategy for identifying close-in-space interactions in RNA molecules. Nucleotides in close three-dimensional proximity are crosslinked with a bi-reactive reagent that covalently links the 2’-hydroxyl groups of the ribose moieties. The identities of crosslinked nucleotides are determined using an engineered reverse transcriptase that jumps across crosslinked sites, resulting in a deletion in the cDNA that is detected using massively parallel sequencing. Here we introduce ShapeJumper, a bioinformatics pipeline to process SHAPE-JuMP sequencing data and to accurately identify through-space interactions, as observed in complex JuMP datasets. ShapeJumper identifies proximal interactions with near-nucleotide resolution using an alignment strategy that is optimized to tolerate the unique non-templated reverse-transcription profile of the engineered crosslink-traversing reverse-transcriptase. JuMP-inspired strategies are now poised to replace adapter-ligation for detecting RNA-RNA interactions in most crosslinking experiments.


2021 ◽  
Author(s):  
Inswasti Cahyani ◽  
John Tyson ◽  
Nadine Holmes ◽  
Josh Quick ◽  
Nicholas Loman ◽  
...  

This sub-protocol is designed to prepare library from extracted ultra-high molecular weight (UHMW) DNA to obtain ultra-long (UL) reads on Nanopore sequencers. The UL library protocol we tested here is based on ONT's rapid kit, i.e., SQK-RAD004, a transposase based adapter ligation kit. This Modified RAD004 protocol consistently produced N50 > 100 kb from a good input quality of UHMW DNA, for when ULK001 is not accessible/available. Transposase-based reaction is done in a large volume of up to 1 ml. The working principle of the RAD004 kit is shown in the diagram below:


2021 ◽  
Author(s):  
John Tyson ◽  
Inswasti Cahyani ◽  
Nadine Holmes ◽  
Josh Quick ◽  
Nicholas Loman ◽  
...  

This sub-protocol is designed to prepare library from extracted ultra-high molecular weight (UHMW) DNA to obtain ultra-long (UL) reads on Nanopore sequencers. The UL library protocol we tested here is based on ONT's rapid kit, i.e., SQK-RAD004, a transposase based adapter ligation kit. We named this protocol KrazyStarFish (KSF). It offers a different approach to UL library prep, by using filter paper shaped as a starfish at the DNA precipitation/clean-up step. It can consistently produced N50 > 100 kb with the right transposase to DNA ratio. This protocol was developed by John Tyson at UBC, Vancouver.


2021 ◽  
Author(s):  
Inswasti Cahyani ◽  
John Tyson ◽  
Nadine Holmes ◽  
Josh Quick ◽  
Nicholas Loman ◽  
...  

This sub-protocol is designed to prepare library from extracted ultra-high molecular weight (UHMW) DNA to obtain ultra-long (UL) reads on Nanopore sequencers. The UL library protocols we tested are based on ONT's rapid kit, i.e., SQK-ULK001, a transposase based adapter ligation kit. Modified ULK001 protocol consistently produced N50 > 100 kb from a good input quality of UHMW DNA and is our recommended route for best output as it is also the most-cost effective. Transposase-based reaction is done in a large volume of up to 1 ml. The working principle of the ULK001 protocol is shown in the diagram below:


2021 ◽  
Author(s):  
Jordan P Cuff ◽  
HooperAS not provided ◽  
Shrinivas Nivrutti Dighe ◽  
Angela Marchbank ◽  
Peter Kille

Nanopore sequencing facilitates the rapid and cost-effective sequencing of long fragment DNA for a massive range of applications. When looking to holistically analyse low-yield DNA samples using nanopore sequencing, the optimal method is likely to involve the PCR Barcoding Kit. This effectively involves blunt end ligation of priming sites onto all extant DNA for holistic amplification to achieve yields suitable for nanopore sequencing. The currently available kits from nanopore facilitate the multiplexing of 96 samples in one sequencing run using this method, but the reagent costs are inherently multiplicative. This protocol is designed to streamline (in terms of cost, reagents and time) the process of sequencing up to 96 samples of genomic DNA through nanopore sequencing. This protocol is best applied to large numbers of samples (up to 96). For smaller numbers of samples, consider the smaller "PCR Barcoding" kits provided by nanopore which similarly achieve holistic DNA amplification and sequencing, but without the need for additional adapter ligation. The protocol is best suited to samples with low DNA yields (100 ng input is recommended). If you can input 1000 ng of DNA from each of your samples, consider using the 96-well Ligation Kit from Oxford Nanopore which can similarly be streamlined in terms of cost and time, but avoids the amplification step.


2021 ◽  
Author(s):  
Thomas W Christy ◽  
Catherine A Giannetti ◽  
Alain Laederach ◽  
Kevin M Weeks

SHAPE-JuMP is a concise strategy for identifying close-in-space interactions in RNA molecules. Nucleotides in close three-dimensional proximity are crosslinked with a bi-reactive reagent that covalently links the 2'-hydroxyl groups of the ribose moieties. The identities of crosslinked nucleotides are determined using an engineered reverse transcriptase that jumps across crosslinked sites, resulting in a deletion in the cDNA that is detected using massively parallel sequencing. Here we introduce ShapeJumper, a bioinformatics pipeline to process SHAPE-JuMP sequencing data and to accurately identify through-space interactions. ShapeJumper identifies proximal interactions with near-nucleotide resolution using an alignment strategy that is optimized to tolerate the unique non-templated reverse-transcription profile of the engineered crosslink-traversing reverse-transcriptase. JuMP-inspired strategies are now poised to replace adapter-ligation for detecting RNA-RNA interactions in most crosslinking experiments.


2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Heming Wang ◽  
Rong Huang ◽  
Ling Li ◽  
Junjin Zhu ◽  
Zhihong Li ◽  
...  

AbstractHigh-throughput sequencing reveals the complex landscape of small noncoding RNAs (sRNAs). However, it is limited by requiring 5′-monophosphate and 3′-hydroxyl in RNAs for adapter ligation and hindered by methylated nucleosides that interfere with reverse transcription. Here we develop Cap-Clip acid pyrophosphatase (Cap-Clip), T4 polynucleotide kinase (PNK) and AlkB/AlkB(D135S)-facilitated small ncRNA sequencing (CPA-seq) to detect and quantify sRNAs with terminus multiplicities and nucleoside methylations. CPA-seq identified a large number of previously undetected sRNAs. Comparison of sRNAs with or without AlkB/AlkB(D135S) treatment reveals nucleoside methylations on sRNAs. Using CPA-seq, we profiled the sRNA transcriptomes (sRNomes) of nine mouse tissues and reported the extensive tissue-specific differences of sRNAs. We also observed the transition of sRNomes during hepatic reprogramming. Knockdown of mesenchymal stem cell-enriched U1-5′ snsRNA promoted hepatic reprogramming. CPA-seq is a powerful tool with high sensitivity and specificity for profiling sRNAs with methylated nucleosides and diverse termini.


Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 278
Author(s):  
Yuri Motorin ◽  
Virginie Marchand

The precise mapping and quantification of the numerous RNA modifications that are present in tRNAs, rRNAs, ncRNAs/miRNAs, and mRNAs remain a major challenge and a top priority of the epitranscriptomics field. After the keystone discoveries of massive m6A methylation in mRNAs, dozens of deep sequencing-based methods and protocols were proposed for the analysis of various RNA modifications, allowing us to considerably extend the list of detectable modified residues. Many of the currently used methods rely on the particular reverse transcription signatures left by RNA modifications in cDNA; these signatures may be naturally present or induced by an appropriate enzymatic or chemical treatment. The newest approaches also include labeling at RNA abasic sites that result from the selective removal of RNA modification or the enhanced cleavage of the RNA ribose-phosphate chain (perhaps also protection from cleavage), followed by specific adapter ligation. Classical affinity/immunoprecipitation-based protocols use either antibodies against modified RNA bases or proteins/enzymes, recognizing RNA modifications. In this survey, we review the most recent achievements in this highly dynamic field, including promising attempts to map RNA modifications by the direct single-molecule sequencing of RNA by nanopores.


protocols.io ◽  
2020 ◽  
Author(s):  
Eric L. Van Nostrand ◽  
Thai B. Nguyen ◽  
Chelsea Gelboin-Burkhart ◽  
Ruth Wang ◽  
Steven M. Blue ◽  
...  
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