Abstract 2286: Use of a synthetic spike-in ladder to measure NGS library complexity

2021 ◽  
Author(s):  
Erin L. Crawford ◽  
Tian Chen ◽  
Daniel J. Craig ◽  
James C. Willey
Keyword(s):  
Library Complexity

scholarly journals Perpustakaan sebagai Teropong Profesionalisme Pustakawan

2018 ◽  
Vol 2 (1) ◽  
pp. 71
Author(s):  
Aris Nurohman
Keyword(s):  
Management Service ◽  
Library Complexity ◽  
And Control

Library is an institution that can not be separated from the role of librarians. Library dependence to the librarian is seen almost in the whole library complexity. Starting from planning, organizing, leadership, and control. Through this writing the authors will contribute a more real attachment between librarians and libraries. Based on some literature proves that between librarian and library can not stand alone. Starting from the management, service products and aspects of library accreditation assessment, librarians are instrumental in contributing enormously. Because of this dependence illustrates that the quality of the library will be seen from the quality of the librarian profession.

scholarly journals A High-Throughput Single-Clone Phage Fluorescence Microwell Immunoassay and Laser-Driven Clonal Retrieval System

Biomolecules ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 517
Author(s):  
Seohee Chang ◽  
Soohyun Kim ◽  
Jerome Han ◽  
Suji Ha ◽  
Hyunho Lee ◽  
...  
Keyword(s):  
High Throughput ◽  
Retrieval System ◽  
Low Cost ◽  
Therapeutic Antibodies ◽  
Amino Acid Residues ◽  
Single Chain ◽  
Scfv Library ◽  
Single Clone ◽  
Library Complexity ◽  
Lower Complexity

Phage display is one of the most frequently used platform technologies utilized to screen and select therapeutic antibodies, and has contributed to the development of more than 10 therapeutic antibodies used in the clinic. Despite advantages like efficiency and low cost, it has intrinsic technical limitations, such as the asymmetrical amplification of the library after each round of biopanning, which is regarded as a reason for it yielding a very limited number of antigen binders. In this study, we developed a high-throughput single-clonal screening system comprised of fluorescence immunoassays and a laser-driven clonal DNA retrieval system using microchip technology. Using this system, from a single-chain variable fragment (scFv) library displayed on phages with a complexity of 5.21 × 105 harboring random mutations at five amino acid residues, more than 70,000 clones—corresponding to ~14% of the library complexity—were screened, resulting in 78 antigen-reactive scFv sequences with mutations restricted to the randomized residues. Our results demonstrate that this system can significantly reduce the number of biopanning rounds, or even eliminate the need for this process for libraries with lower complexity, providing an opportunity to obtain more diverse clones from the library.

scholarly journals PEPPRO: quality control and processing of nascent RNA profiling data

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Jason P. Smith ◽  
Arun B. Dutta ◽  
Kizhakke Mattada Sathyan ◽  
Michael J. Guertin ◽  
Nathan C. Sheffield
Keyword(s):  
Fault Tolerant ◽  
Analysis Pipeline ◽  
Web Based ◽  
Rna Integrity ◽  
Rna Profiling ◽  
Project Report ◽  
Library Complexity ◽  
Nascent Rna ◽  
Assess Quality ◽  
Downstream Analysis

AbstractNascent RNA profiling is growing in popularity; however, there is no standard analysis pipeline to uniformly process the data and assess quality. Here, we introduce PEPPRO, a comprehensive, scalable workflow for GRO-seq, PRO-seq, and ChRO-seq data. PEPPRO produces uniformly processed output files for downstream analysis and assesses adapter abundance, RNA integrity, library complexity, nascent RNA purity, and run-on efficiency. PEPPRO is restartable and fault-tolerant, records copious logs, and provides a web-based project report. PEPPRO can be run locally or using a cluster, providing a portable first step for genomic nascent RNA analysis.

scholarly journals Quality control and processing of nascent RNA profiling data

2020 ◽  
Author(s):  
Jason P. Smith ◽  
Arun B. Dutta ◽  
Kizhakke Mattada Sathyan ◽  
Michael J. Guertin ◽  
Nathan C. Sheffield
Keyword(s):  
Quality Control ◽  
Fault Tolerant ◽  
Resource Manager ◽  
Web Based ◽  
Rna Integrity ◽  
Rna Profiling ◽  
Library Complexity ◽  
Nascent Rna ◽  
Assess Quality ◽  
Downstream Analysis

Experiments that profile nascent RNA are growing in popularity; however, there is no standard analysis pipeline to uniformly process the data and assess quality. Here, we introduce PEPPRO, a comprehensive, scalable workflow for GRO-seq, PRO-seq, and ChRO-seq data. PEPPRO produces uniform processed output files for downstream analysis, including alignment files, signal tracks, and count matrices. Furthermore, PEPPRO simplifies downstream analysis by using a standard project definition format which can be read using metadata APIs in R and Python. For quality control, PEPPRO provides several novel statistics and plots, including assessments of adapter abundance, RNA integrity, library complexity, nascent RNA purity, and run-on efficiency. PEPPRO is restartable and fault-tolerant, records copious logs, and provides a web-based project report for navigating results. It can be run on local hardware or using any cluster resource manager, using either native software or a provided modular Linux container environment. PEPPRO is thus a robust and portable first step for genomic nascent RNA analysis.AvailabilityBSD2-licensed code and documentation: https://peppro.databio.org.

scholarly journals Massively parallel enzyme kinetics reveals the substrate recognition landscape of the metalloprotease ADAMTS13

2015 ◽  
Vol 112 (30) ◽  
pp. 9328-9333 ◽  
Author(s):  
Colin A. Kretz ◽  
Manhong Dai ◽  
Onuralp Soylemez ◽  
Andrew Yee ◽  
Karl C. Desch ◽  
...  
Keyword(s):  
Amino Acid ◽  
Enzyme Kinetics ◽  
High Throughput Sequencing ◽  
Phage Display Library ◽  
Massively Parallel ◽  
Single Amino Acid ◽  
Reaction Rate Constants ◽  
Binding Domains ◽  
Dependent Change ◽  
Library Complexity

Proteases play important roles in many biologic processes and are key mediators of cancer, inflammation, and thrombosis. However, comprehensive and quantitative techniques to define the substrate specificity profile of proteases are lacking. The metalloprotease ADAMTS13 regulates blood coagulation by cleaving von Willebrand factor (VWF), reducing its procoagulant activity. A mutagenized substrate phage display library based on a 73-amino acid fragment of VWF was constructed, and the ADAMTS13-dependent change in library complexity was evaluated over reaction time points, using high-throughput sequencing. Reaction rate constants (kcat/KM) were calculated for nearly every possible single amino acid substitution within this fragment. This massively parallel enzyme kinetics analysis detailed the specificity of ADAMTS13 and demonstrated the critical importance of the P1-P1′ substrate residues while defining exosite binding domains. These data provided empirical evidence for the propensity for epistasis within VWF and showed strong correlation to conservation across orthologs, highlighting evolutionary selective pressures for VWF.

scholarly journals Prime-seq, efficient and powerful bulk RNA-sequencing

2021 ◽  
Author(s):  
Aleksandar Janjic ◽  
Lucas Esteban Wange ◽  
Johannes Walter Bagnoli ◽  
Johanna Geuder ◽  
Phong Nguyen ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Statistical Power ◽  
Gene Expression Analysis ◽  
Measurement Accuracy ◽  
Rna Isolation ◽  
Rna Seq ◽  
Specialized Equipment ◽  
Library Complexity ◽  
Cost Efficient ◽  
Generation Sequencing

With the advent of Next Generation Sequencing, RNA-sequencing (RNA-seq) has become the major method for quantitative gene expression analysis. Reducing library costs by early barcoding has propelled single-cell RNA-seq, but has not yet caught on for bulk RNA-seq. Here, we optimized and validated a bulk RNA-seq method we call prime-seq. We show that with respect to library complexity, measurement accuracy, and statistical power it performs equivalent to TruSeq, a standard bulk RNA-seq method, but is four-fold more cost-efficient due to almost 50-fold cheaper library costs. We also validate a direct RNA isolation step that further improves cost and time-efficiency, show that intronic reads are derived from RNA, validate that prime-seq performs optimal with only 1,000 cells as input, and calculate that prime-seq is the most cost-efficient bulk RNA-seq method currently available. We discuss why many labs would profit from a cost-efficient early barcoding RNA-seq protocol and argue that prime-seq is well suited for setting up such a protocol as it is well validated, well documented, and requires no specialized equipment.

scholarly journals Transposase-assisted tagmentation of RNA/DNA hybrid duplexes

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Bo Lu ◽  
Liting Dong ◽  
Danyang Yi ◽  
Meiling Zhang ◽  
Chenxu Zhu ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Differential Expression Analysis ◽  
Cost Effective ◽  
Gene Expression Measurement ◽  
Rna Seq ◽  
Library Construction ◽  
Rna Biology ◽  
Library Complexity ◽  
Tn5 Transposase

Tn5-mediated transposition of double-strand DNA has been widely utilized in various high-throughput sequencing applications. Here, we report that the Tn5 transposase is also capable of direct tagmentation of RNA/DNA hybrids in vitro. As a proof-of-concept application, we utilized this activity to replace the traditional library construction procedure of RNA sequencing, which contains many laborious and time-consuming processes. Results of Transposase-assisted RNA/DNA hybrids Co-tagmEntation (termed ‘TRACE-seq’) are compared to traditional RNA-seq methods in terms of detected gene number, gene body coverage, gene expression measurement, library complexity, and differential expression analysis. At the meantime, TRACE-seq enables a cost-effective one-tube library construction protocol and hence is more rapid (within 6 hr) and convenient. We expect this tagmentation activity on RNA/DNA hybrids to have broad potentials on RNA biology and chromatin research.

scholarly journals Multiplexed pooled library screening with Cpf1

2018 ◽  
Author(s):  
Jintan Liu ◽  
Sanjana Srinivasan ◽  
Chieh-Yuan Li ◽  
I-Lin Ho ◽  
Gang Wang ◽  
...  
Keyword(s):  
Rna Interference ◽  
Gene Targeting ◽  
High Throughput Screening ◽  
Screening Strategy ◽  
Whole Genome ◽  
Library Screening ◽  
Library Size ◽  
Trade Off ◽  
Knock Out ◽  
Library Complexity

AbstractRNA interference and CRISPR/Cas9-based pooled library screens have revolutionized the field of functional genomics. However, currently available pooled library screens face a trade-off between library effectiveness and library complexity. We developed a multiplexed, high-throughput screening strategy based on an optimized AsCpf1 nuclease that minimizes library size without sacrificing gene targeting efficiency. Our AsCpf1-based multiplexed library performed similarly well compared to currently available CRISPR/Cas9 libraries, but with a single polycistronic crRNA clone targeting each gene. With this strategy, we constructed the smallest whole-genome knock-out library available, “Mini-human” for the human genome, which is one-fourth the size of the smallest CRISPR library currently available.

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