scholarly journals Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

10.3791/56837 ◽  
2018 ◽  
Author(s):  
Saman Saeidi ◽  
Michael R. McKain ◽  
Elizabeth A. Kellogg
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Dna Isolation ◽  
Herbarium Specimens ◽  
Library Construction ◽  
Sequencing Library

scholarly journals Small RNA Expression Profiling by High-Throughput Sequencing: Implications of Enzymatic Manipulation

2012 ◽  
Vol 2012 ◽  
pp. 1-15 ◽  
Author(s):  
Fanglei Zhuang ◽  
Ryan T. Fuchs ◽  
G. Brett Robb
Keyword(s):  
High Throughput ◽  
Expression Profiling ◽  
Messenger Rna ◽  
High Throughput Sequencing ◽  
Biological Processes ◽  
Cellular Processes ◽  
Disease States ◽  
Powerful Approach ◽  
Sequencing Library ◽  
Rna Expression Profiling

Eukaryotic regulatory small RNAs (sRNAs) play significant roles in many fundamental cellular processes. As such, they have emerged as useful biomarkers for diseases and cell differentiation states. sRNA-based biomarkers outperform traditional messenger RNA-based biomarkers by testing fewer targets with greater accuracy and providing earlier detection for disease states. Therefore, expression profiling of sRNAs is fundamentally important to further advance the understanding of biological processes, as well as diagnosis and treatment of diseases. High-throughput sequencing (HTS) is a powerful approach for both sRNA discovery and expression profiling. Here, we discuss the general considerations for sRNA-based HTS profiling methods from RNA preparation to sequencing library construction, with a focus on the causes of systematic error. By examining the enzymatic manipulation steps of sRNA expression profiling, this paper aims to demystify current HTS-based sRNA profiling approaches and to aid researchers in the informed design and interpretation of profiling experiments.

scholarly journals DNA extraction and sequencing of the Mawangdui ancient cadaver protected by formalin

2020 ◽  
Author(s):  
Hua-Lin Huang ◽  
Shikui Yin ◽  
Huifang Zhao ◽  
Chao Tian ◽  
Jufang Huang ◽  
...  
Keyword(s):  
Dna Extraction ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Pcr Amplification ◽  
Magnetic Bead ◽  
Sequencing Data ◽  
Sequencing Library ◽  
Bead Method ◽  
Formalin Fixed ◽  
Better Than

AbstractMawangdui ancient Cadaver is the first wet corpse found in the world, which is famous for being immortal for over two thousands of years. After being unearthed, the female corpse was immersed in the formalin protective solution for more than 40 years. We used magnetic bead method and formalin fixed paraffing (FFPE) method to extract the DNA of the female corpse, respectively. PCR amplification, sanger sequencing, library building, high throughput sequencing (testing) and data processing were carried out on the DNA samples, and about 0.5% of the whole genome coverage sequencing data was obtained. Comparing the results of DNA trough two extraction and sequencing methods. We found that the FFPE and high throughput sequencing methods is better than others for DNA extraction of the ancient samples which were preserved in formalin, providing a guidance for dealing with formalin preserved ancient samples in the future.

scholarly journals FLASH: ultra-fast protocol to identify RNA–protein interactions in cells

2019 ◽  
Vol 48 (3) ◽  
pp. e15-e15 ◽  
Author(s):  
Ibrahim Avsar Ilik ◽  
Tugce Aktas ◽  
Daniel Maticzka ◽  
Rolf Backofen ◽  
Asifa Akhtar
Keyword(s):  
High Throughput ◽  
Protein Interactions ◽  
Binding Sites ◽  
Binding Proteins ◽  
High Throughput Sequencing ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Affinity Purification ◽  
Sequencing Library

Abstract Determination of the in vivo binding sites of RNA-binding proteins (RBPs) is paramount to understanding their function and how they affect different aspects of gene regulation. With hundreds of RNA-binding proteins identified in human cells, a flexible, high-resolution, high-throughput, highly multiplexible and radioactivity-free method to determine their binding sites has not been described to date. Here we report FLASH (Fast Ligation of RNA after some sort of Affinity Purification for High-throughput Sequencing), which uses a special adapter design and an optimized protocol to determine protein–RNA interactions in living cells. The entire FLASH protocol, starting from cells on plates to a sequencing library, takes 1.5 days. We demonstrate the flexibility, speed and versatility of FLASH by using it to determine RNA targets of both tagged and endogenously expressed proteins under diverse conditions in vivo.

scholarly journals Reply to Grigoriev et al., “Sequences of SARS-CoV-2 “Hybrids” with the Human Genome: Signs 1 of Non-coding RNA?”

2021 ◽  
Author(s):  
Bingyu Yan ◽  
Srishti Chakravorty ◽  
Carmen Mirabelli ◽  
Luopin Wang ◽  
Jorge L. Trujillo-Ochoa ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Host Cells ◽  
Viral Gene ◽  
Gene Expressions ◽  
Biologically Relevant ◽  
Sequencing Library ◽  
Infected Cells ◽  
Template Switch ◽  
Host Virus Interactions

High throughput sequencing reads from virally infected cells provide detailed information about both the infected host cells and invading viruses (1). For example, RNA-sequencing techniques from infected cells contains reads that unequivocally align to either the host or the viral transcriptomes, enabling quantification of host and viral gene expressions (2). Occasionally, there are reads with split characteristics, having one part (e.g., the 5’ end) unambiguously matching the host and another part (e.g., the 3’ end) clearly matching the viral genomes. The split characteristic with unambiguous matching on either part is the key here, typically requiring convincing stretches of sequence matches such as >30bp that we used in our analysis (3). Such reads are termed host-virus chimeric reads (HVCRs). Indeed, HVCRs that surpass statistical reproducibility and signal-to-noise standards might carry novel insights into the biology of host-virus interactions (4, 5). Thus, it is important to unambiguously detect statistically rigorous and biologically relevant HVCRs. We and others have shown that detection of relevant HVCRs is complicated by unfaithful reverse-transcriptase and polymerase enzymes that template-switch during typical high throughput sequencing library preparation protocols (6–9).

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