3' RNA Adapter Ligation to Input RNA v1

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.

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Targeted nanopore sequencing with Cas9-guided adapter ligation

Nature Biotechnology ◽

10.1038/s41587-020-0407-5 ◽

2020 ◽

Vol 38 (4) ◽

pp. 433-438 ◽

Cited By ~ 35

Author(s):

Timothy Gilpatrick ◽

Isac Lee ◽

James E. Graham ◽

Etienne Raimondeau ◽

Rebecca Bowen ◽

...

Keyword(s):

Nanopore Sequencing ◽

Adapter Ligation

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Preparation of Amplicon Libraries for Metabarcoding of Marine Eukaryotes Using Illumina MiSeq: The Adapter Ligation Method

Methods in Molecular Biology - Marine Genomics ◽

10.1007/978-1-4939-3774-5_14 ◽

2016 ◽

pp. 209-218 ◽

Cited By ~ 7

Author(s):

Matthieu Leray ◽

Quiterie Haenel ◽

Sarah J. Bourlat

Keyword(s):

Illumina Miseq ◽

Adapter Ligation ◽

Ligation Method

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PacBio library preparation using blunt-end adapter ligation produces significant artefactual fusion DNA sequences

10.1101/245241 ◽

2018 ◽

Author(s):

Paul Griffith ◽

Castle Raley ◽

David Sun ◽

Yongmei Zhao ◽

Zhonghe Sun ◽

...

Keyword(s):

Single Molecule ◽

Dna Sequences ◽

Turnaround Time ◽

Library Preparation ◽

Adapter Ligation ◽

Sequence Production ◽

Long Reads ◽

Complex Structural Variation ◽

Long Read ◽

Complex Structural

AbstractPacific Biosciences’ (PacBio) RS II sequencer, utilizing Single-Molecule, Real-Time (SMRT) technology, has revolutionized next-generation sequencing by providing an accurate long-read platform. PacBio single-molecule long reads have been used to delineate complex spliceoforms, detect mutations in highly homologous sequences, identify mRNA chimeras and chromosomal translocations, accurately haplotype phasing over multiple kilobase distances and aid in assembly of genomes with complex structural variation. The PacBio protocol for preparation of sequencing templates employs blunt-end hairpin adapter ligation, which enables a short turnaround time for sequence production. However, we have found a significant portion of sequencing yield contains chimeric reads resulting from blunt-end ligation of multiple template molecules to each other prior to adapter ligation. These artefactual fusion DNA sequences pose a major challenge to analysis and can lead to false-positive detection of fusion events. We assessed the frequency of artefactual fusion when using blunt-end adapter ligation and compared it to an alternative method using A/T overhang adapter ligation. The A/T overhang adapter ligation method showed a vast improvement in limiting artefactual fusion events and is now our recommended procedure for adapter ligation during PacBio library preparation.

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Excess primer degradation by Exo I improves the preparation of 3′ cDNA ligation-based sequencing libraries

BioTechniques ◽

10.2144/btn-2018-0178 ◽

2019 ◽

Vol 67 (3) ◽

pp. 110-116

Author(s):

Christel Hougård Enroth ◽

Annaleigh Ohrt Fehler ◽

Line Dahl Poulsen ◽

Jeppe Vinther

Keyword(s):

Escherichia Coli ◽

Rna Sequencing ◽

Small Rna ◽

Reverse Transcription ◽

Library Construction ◽

Reverse Transcription Primer ◽

Adapter Ligation ◽

Sequencing Library

RNA sequencing library construction using single-stranded ligation of a DNA adapter to 3′ ends of cDNAs often produces primer–adapter byproducts, which compete with cDNA–adapter ligation products during library amplification and, therefore, reduces the number of informative sequencing reads. We find that Escherichia coli Exo I digestion efficiently and selectively removes surplus reverse transcription primer and thereby reduces the primer–adapter product contamination in 3′ cDNA ligation-based sequencing libraries, including small RNA libraries, which are typically similar in size to the primer–adapter products. We further demonstrate that Exo I treatment does not lead to trimming of the cDNA 3′ end when duplexed with the RNA template. Exo I digestion is easy to perform and implement in other protocols and could facilitate a more widespread use of 3′ cDNA ligation for sequencing-based applications.

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FindingNemo Library 1: Modified ULK001 v1

10.17504/protocols.io.bxhhpj36 ◽

2021 ◽

Author(s):

Inswasti Cahyani ◽

John Tyson ◽

Nadine Holmes ◽

Josh Quick ◽

Nicholas Loman ◽

...

Keyword(s):

Molecular Weight ◽

High Molecular Weight ◽

Large Volume ◽

Cost Effective ◽

Input Quality ◽

Adapter Ligation ◽

Working Principle ◽

Ultra High Molecular Weight

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:

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A streamlined protocol for high-throughput amplification-based analysis of DNA samples via nanopore sequencing (based on the 96-well PCR barcoding kit) v1

10.17504/protocols.io.bw2cpgaw ◽

2021 ◽

Author(s):

Jordan P Cuff ◽

HooperAS not provided ◽

Shrinivas Nivrutti Dighe ◽

Angela Marchbank ◽

Peter Kille

Keyword(s):

High Throughput ◽

Genomic Dna ◽

Dna Amplification ◽

Cost Effective ◽

Nanopore Sequencing ◽

Optimal Method ◽

Adapter Ligation ◽

Large Numbers ◽

Oxford Nanopore

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.

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Analysis of RNA Modifications by Second- and Third-Generation Deep Sequencing: 2020 Update

Genes ◽

10.3390/genes12020278 ◽

2021 ◽

Vol 12 (2) ◽

pp. 278

Author(s):

Yuri Motorin ◽

Virginie Marchand

Keyword(s):

Single Molecule ◽

Deep Sequencing ◽

Rna Modification ◽

Rna Modifications ◽

Single Molecule Sequencing ◽

Abasic Sites ◽

Phosphate Chain ◽

Adapter Ligation ◽

Ribose Phosphate ◽

Precise Mapping

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.

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