Microarray Generation of Thousand-Member Oligonucleotide Libraries

Svensen, Nina; Díaz‐Mochón, Juan José; Bradley, Mark

doi:10.1371/journal.pone.0024906

Cited by 12 publications

(8 citation statements)

References 43 publications

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“…As an example, the measured signal for a given target in traditional RNA-sequencing is filtered through a set of biases such as GC content, transcript length, transcript fragmentation efficiency during library preparation, ligation of sequencing adapters and so forth. These systematic effects can be large at times [33] . In this inter-platform comparison we sought to demonstrate that once these large differences were corrected for, both our method as well as TaqMan qPCR and traditional RNA-sequencing will be largely concordant for absolute transcript abundance measurements in Samples C and D. Based on these pairwise comparisons, we observed a high degree of cross-platform agreement between competitive amplicon library preparation targeted RNA-sequencing with TaqMan qPCR (R 2 = 0.96), as well as Illumina RNA-Sequencing (R 2 = 0.94) for absolute expression measurements.…”

Section: Resultsmentioning

confidence: 99%

“…competitive amplicon library preparation targeted RNA-sequencing vs. TaqMan vs. traditional RNA-sequencing on Illumina NGS) was largely systematic for each target ( Figures S1 , S2 ). One source of systematic variation between methods may be ligation bias exhibited during more traditional RNA-sequencing methods, which can be as large as 1000-fold [33] . One possible source of non-systematic inter-platform variation in measurement of a select number of endogenous assay targets is that different transcript isoforms were assessed by the different platforms.…”

Section: Discussionmentioning

confidence: 99%

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Targeted RNA-Sequencing with Competitive Multiplex-PCR Amplicon Libraries

et al. 2013

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Whole transcriptome RNA-sequencing is a powerful tool, but is costly and yields complex data sets that limit its utility in molecular diagnostic testing. A targeted quantitative RNA-sequencing method that is reproducible and reduces the number of sequencing reads required to measure transcripts over the full range of expression would be better suited to diagnostic testing. Toward this goal, we developed a competitive multiplex PCR-based amplicon sequencing library preparation method that a) targets only the sequences of interest and b) controls for inter-target variation in PCR amplification during library preparation by measuring each transcript native template relative to a known number of synthetic competitive template internal standard copies. To determine the utility of this method, we intentionally selected PCR conditions that would cause transcript amplification products (amplicons) to converge toward equimolar concentrations (normalization) during library preparation. We then tested whether this approach would enable accurate and reproducible quantification of each transcript across multiple library preparations, and at the same time reduce (through normalization) total sequencing reads required for quantification of transcript targets across a large range of expression. We demonstrate excellent reproducibility (R2 = 0.997) with 97% accuracy to detect 2-fold change using External RNA Controls Consortium (ERCC) reference materials; high inter-day, inter-site and inter-library concordance (R2 = 0.97–0.99) using FDA Sequencing Quality Control (SEQC) reference materials; and cross-platform concordance with both TaqMan qPCR (R2 = 0.96) and whole transcriptome RNA-sequencing following “traditional” library preparation using Illumina NGS kits (R2 = 0.94). Using this method, sequencing reads required to accurately quantify more than 100 targeted transcripts expressed over a 107-fold range was reduced more than 10,000-fold, from 2.3×109 to 1.4×105 sequencing reads. These studies demonstrate that the competitive multiplex-PCR amplicon library preparation method presented here provides the quality control, reproducibility, and reduced sequencing reads necessary for development and implementation of targeted quantitative RNA-sequencing biomarkers in molecular diagnostic testing.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Targeted RNA-Sequencing with Competitive Multiplex-PCR Amplicon Libraries

et al. 2013

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“…Together these features make DECKO not only practical for knockout of individual sites, but for screening approaches that require multiplex cloning of many target constructs in a pooled format [ 23 , 41 , 42 ]. The array-based megasynthesis required to produce such oligonucleotide libraries are capable of generating pools at the 165 bp length required by DECKO [ 23 , 25 , 43 ].…”

Section: Discussionmentioning

confidence: 99%

DECKO: Single-oligo, dual-CRISPR deletion of genomic elements including long non-coding RNAs

et al. 2015

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BackgroundCRISPR genome-editing technology makes it possible to quickly and cheaply delete non-protein-coding regulatory elements. We present a vector system adapted for this purpose called DECKO (Double Excision CRISPR Knockout), which applies a simple two-step cloning to generate lentiviral vectors expressing two guide RNAs (gRNAs) simultaneously. The key feature of DECKO is its use of a single 165 bp starting oligonucleotide carrying the variable sequences of both gRNAs, making it fully scalable from single-locus studies to complex library cloning.ResultsWe apply DECKO to deleting the promoters of one protein-coding gene and two oncogenic lncRNAs, UCA1 and the highly-expressed MALAT1, focus of many previous studies employing RNA interference approaches. DECKO successfully deleted genomic fragments ranging in size from 100 to 3000 bp in four human cell lines. Using a clone-derivation workflow lasting approximately 20 days, we obtained 9 homozygous and 17 heterozygous promoter knockouts in three human cell lines. Frequent target region inversions were observed. These clones have reductions in steady-state MALAT1 RNA levels of up to 98 % and display reduced proliferation rates.ConclusionsWe present a dual CRISPR tool, DECKO, which is cloned using a single starting oligonucleotide, thereby affording simplicity and scalability to CRISPR knockout studies of non-coding genomic elements, including long non-coding RNAs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2086-z) contains supplementary material, which is available to authorized users.

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“…For example, even two decades ago, Lipshutz reviewed the design and synthesis of high‐density and two‐dimensional arrays of synthetic oligonucleotides such was the sophistication achieved in the combination of GeneChip probe arrays. The DNA array libraries have been used for a broad set of applications such as gene expression monitoring, sequence analysis, and genotyping, quantitative measurement of gene expression . DNA encoded libraries have been efficiently used in drug discovery as well as to discover novel ligands.…”

Section: Introductionmentioning

confidence: 99%

“…The DNA array libraries have been used for a broad set of applications such as gene expression monitoring, sequence analysis, and genotyping, quantitative measurement of gene expression. [26][27][28][29][30][31][32] DNA encoded libraries have been efficiently used in drug discovery [33][34][35] as well as to discover novel ligands.…”

Section: Introductionmentioning

confidence: 99%

Glycosylation on Unprotected or Partially Protected Acceptors

Ding

Prasad²,

Wang

2020

Eur J Org Chem

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Over the last 30 years, there have been several methods developed for the synthesis of oligosaccharides, such as combinatorial synthesis, programmable one‐pot glycosylations, pre‐activation‐based chemo‐selective glycosylation strategy, random glycosylation, chemo‐enzymatic processes, and automated platforms. Distinguishing hydroxyl groups in carbohydrate monomers form the crux of oligosaccharides synthesis. The protection of hydroxyl groups stops glycosylation at undesired positions on carbohydrates, but additional steps are required to install and remove the protecting groups. The targeted functionalization of carbohydrates relies mostly on the protection of hydroxyl groups that are not supposed to undergo glycosylation and to leave the hydroxyl group unprotected where the glycosylation should occur. Numerous procedures to avoid or decrease protecting group manipulations have been developed for the synthesis of a single or an oligosaccharide library, such as glycosylation on unprotected or partially protected acceptors, and random glycosylation on unprotected acceptors. In this review, overall approaches for glycosylation on unprotected or partially protected acceptors in the synthesis of oligosaccharides and oligosaccharide libraries are summarized, the glycosylation on unprotected or partially protected carbohydrate acceptors with or without tin or boron mediation, and random glycosylation on unprotected or partially protected acceptors in solution phase or on solid phase are discussed.

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Microarray Generation of Thousand-Member Oligonucleotide Libraries

Cited by 12 publications

References 43 publications

Targeted RNA-Sequencing with Competitive Multiplex-PCR Amplicon Libraries

Targeted RNA-Sequencing with Competitive Multiplex-PCR Amplicon Libraries

DECKO: Single-oligo, dual-CRISPR deletion of genomic elements including long non-coding RNAs

Glycosylation on Unprotected or Partially Protected Acceptors

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