scholarly journals Optimised protocol of QIAamp® DNA mini Kit for bacteria genomic DNA extraction from both pure and mixture sample

2019 ◽  
Author(s):  
Zhong Yang
Keyword(s):  
Dna Extraction ◽  
Complex Mixture ◽  
Sequencing Technology ◽  
Optimized Protocol ◽  
Next Generation Sequencing Ngs ◽  
Mixture Samples ◽  
Sequencing Result ◽  
Generation Sequencing ◽  
Mixture Sample

Abstract As the sequencing technology developed, Next Generation Sequencing (NGS) has been used by more and more microbiology lab from different fields. Meanwhile , the quality of DNA will highly influence the sequencing result. Recently, there is an increasingly requirement for effective DNA extraction protocol to get high quality DNA from both pure and mixture cultures. Especially for researchers who what to study the microorganisms from complex mixture samples from food and environment. In this protocol, we modified the protocol of a commercial DNA extraction kit from Qiagen, and made it suitable for DNA extraction of different sample sources. The optimized protocol is proved to be effective. The DNA extracted with this protocol has been sent for NGS and get many interesting result.

scholarly journals METODE EKSTRAKSI DNA TANAMAN TANPA PRESIPITASI ETANOL UNTUK KEGIATAN POLYMERASE CHAIN REACTION (PCR)

2019 ◽  
Vol 6 (1) ◽  
pp. 29
Author(s):  
Kristianto Nugroho ◽  
Rerenstradika Tizar Terryana ◽  
. Reflinur ◽  
Puji Lestari
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Extraction ◽  
Simple Sequence Repeat ◽  
Sequence Repeat ◽  
Chain Reaction ◽  
Ethanol Precipitation ◽  
Polymerase Chain ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing ◽  
Simple Sequence

A Simplified Plant DNA Extraction Protocol without Ethanol Precipitation for Polymerase Chain Reaction (PCR) Activities ABSTRACTMolecular-based research in agriculture includes DNA extraction stage involving DNA precipitation using ethanol or isopropanol which tends to take a long time. The purpose of this study was to obtain a plant DNA extraction method for Polymerase Chain Reaction (PCR) activities without going through the ethanol precipitation stage. Five important agricultural commodity crops, namely rice, corn, soybeans, chilies, and shallots were extracted by DNA using the modified Doyle and Doyle method. After the extraction phase using chloroform and isoamil alcohol solvents, the supernatant obtained was not precipitated using ethanol but was directly diluted and used as a template in PCR activities using two pairs of Simple Sequence Repeat (SSR) markers. The results showed that all samples could be well amplified, and amplicon tape visualized in both 1% agarose gel and 6% polyacrylamide gel were clearly visible. This method could save time and material, and reduce the dependence on liquid nitrogen. But this method is still limited to PCR requirements only, and cannot be used for activities that require high quality and quantity of DNA such as Next Generation Sequencing (NGS), digestion, and hybridization.Keywords: DNA extraction, ethanol precipitation, liquid nitrogen, PCR, SSR,  ABSTRAKPenelitian berbasis molekuler pada bidang pertanian mencakup tahapan ekstraksi DNA yang melibatkan presipitasi DNA menggunakan etanol atau isopropanol yang cenderung memakan waktu lama. Tujuan penelitian ini adalah untuk memperoleh metode ekstraksi DNA tanaman untuk kegiatan Polymerase Chain Reaction (PCR) tanpa melalui tahapan presipitasi etanol. Lima tanaman komoditas pertanian penting yaitu padi, jagung, kedelai, cabai, dan bawang merah diekstraksi DNA-nya menggunakan metode Doyle and Doyle yang dimodifikasi. Setelah tahap ekstraksi menggunakan pelarut kloroform dan isoamil alkohol, supernatan yang terbentuk tidak dipresipistasi menggunakan etanol melainkan langsung diencerkan dan digunakan sebagai template dalam kegiatan PCR menggunakan dua pasang marka Simple Sequence Repeat (SSR). Hasil menunjukkan bahwa seluruh sampel dapat teramplifikasi dengan baik serta pita hasil amplikon yang tervisualisasi baik pada gel agarosa 1% maupun gel poliakrilamid 6% terlihat jelas. Metode ini dapat menghemat waktu dan bahan serta mengurangi ketergantungan pemakaian nitrogen cair. Tetapi metode ini masih terbatas hanya untuk kebutuhan PCR saja dan tidak dapat digunakan untuk kegiatan yang membutuhkan DNA dengan kualitas serta kuantitas tinggi seperti Next Generation Sequencing (NGS), digesti, maupun hibridisasi.Kata Kunci: ekstraksi DNA, nitrogen cair, PCR, presipitasi etanol, SSR

scholarly journals Optimized protocol to isolate high quality genomic DNA from different tissues of a palm species

Hoehnea ◽  
2019 ◽  
Vol 46 (2) ◽  
Author(s):  
Marília Souza Lucas ◽  
Carolina da Silva Carvalho ◽  
Giovane Böerner Hypolito ◽  
Marina Corrêa Côrtes
Keyword(s):  
Dna Extraction ◽  
Genomic Dna ◽  
Dna Isolation ◽  
Molecular Techniques ◽  
Tissue Type ◽  
High Quality ◽  
Optimized Protocol ◽  
Palm Species ◽  
Different Tissues

ABSTRACT The application of molecular techniques to tackle ecological and evolutionary questions requires genomic DNA in good quality and quantity. The quality of the isolated DNA, however, can be influenced by the tissue type and the way the sample was conserved and manipulated. Therefore, customizing protocols to improve the DNA isolation and locus amplification is crucial. We optimized a cheap and manual protocol of DNA extraction and microsatellites amplification using five different tissues of a palm species of the brazilian Atlantic Forest. We successfully extracted DNA from all five tissue types. Leaf, stem, and endocarp of non-dispersed seeds presented the highest rates of successful DNA extraction and microsatellite amplification; whereas root, endocarp of dispersed seeds, and embryo showed the lowest quality and quantity. Based on these results, we discussed the implications of using different tissues for studies about seed dispersal, pollination, and population genetics.

scholarly journals Sequencing your genome: your future is here, but are you sure you want to know it?

2014 ◽  
Vol 96 ◽  
Author(s):  
NIR PILLAR ◽  
OFER ISAKOV ◽  
NOAM SHOMRON
Keyword(s):  
Next Generation Sequencing ◽  
Research Laboratory ◽  
High Throughput Sequencing ◽  
Genomic Research ◽  
Sequencing Technology ◽  
Clinical Diagnostic Laboratory ◽  
Diagnostic Laboratory ◽  
The Past ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Next-generation sequencing (NGS; also known as deep sequencing or ultra-high throughput sequencing) has probably been the most important tool for genomic research over the past few years. NGS has led to numerous discoveries and scientific breakthroughs in the genetic field. The sequencing technology that has entered the research laboratory in the past decade is now being introduced into the clinical diagnostic laboratory. Consequently, NGS results are becoming available in the medical arena as abundance of clinically relevant variants, conferring predisposition to disease, are being discovered at a growing rate (Stanley, 2014).

scholarly journals Review of Clinical Next-Generation Sequencing

2017 ◽  
Vol 141 (11) ◽  
pp. 1544-1557 ◽  
Author(s):  
Sophia Yohe ◽  
Bharat Thyagarajan
Keyword(s):  
Next Generation Sequencing ◽  
Clinical Care ◽  
Cost Effective ◽  
Next Generation ◽  
Inherited Disorders ◽  
Academic Center ◽  
Next Generation Sequencing Ngs ◽  
Ngs Data ◽  
Generation Sequencing

Context.— Next-generation sequencing (NGS) is a technology being used by many laboratories to test for inherited disorders and tumor mutations. This technology is new for many practicing pathologists, who may not be familiar with the uses, methodology, and limitations of NGS. Objective.— To familiarize pathologists with several aspects of NGS, including current and expanding uses; methodology including wet bench aspects, bioinformatics, and interpretation; validation and proficiency; limitations; and issues related to the integration of NGS data into patient care. Data Sources.— The review is based on peer-reviewed literature and personal experience using NGS in a clinical setting at a major academic center. Conclusions.— The clinical applications of NGS will increase as the technology, bioinformatics, and resources evolve to address the limitations and improve quality of results. The challenge for clinical laboratories is to ensure testing is clinically relevant, cost-effective, and can be integrated into clinical care.

scholarly journals DNA extraction from resin producing Boswellia tree

2019 ◽  
Author(s):  
Adil Khan ◽  
Abdul Latif Khan ◽  
Sajjad Asaf ◽  
Ahmed Al-Harrasi ◽  
Ahmed Al-Rawahi
Keyword(s):  
Cetyl Trimethyl Ammonium Bromide ◽  
Ammonium Bromide ◽  
High Quality ◽  
Commiphora Wightii ◽  
Optimized Protocol ◽  
Handling Methods ◽  
Cetyl Trimethyl Ammonium ◽  
Passiflora Foetida ◽  
Generation Sequencing

Abstract Wild and medicinal plants producing resin and ladle with phenolic and polysaccharides have been found a major hurdle to extract high quality of genomic DNA. These contaminants co -precipitate with DNA and inhibit enzymatic modification of DNA. Previously reported protocols yielded highly viscous DNA regimen that were not amicable to down-stream analysis. We tried several commonly used protocols but were unable to isolate high quality DNA from frankincense producing Boswellia sacra. We extensively optimized the Cetyl Trimethyl Ammonium Bromide (CTAB) based protocol used for Commiphora wightii and Passiflora foetida previously. The current methos involves changes in buffer conditions, handling methods, and cycles of steps to remove resin at earlier stages of extracton. The obtained gDNA can be used for genetic diversity, DNA barcoding and next generation sequencing approaches. To our knowledge, this is the first optimized protocol which a rapid and less laborious for the extraction of DNA from Boswellia species.

scholarly journals The Evolving Landscape of Chronic Lymphocytic Leukemia on Diagnosis, Prognosis and Treatment

Diagnostics ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 853
Author(s):  
Claudia Pérez-Carretero ◽  
Isabel González-Gascón-y-Marín ◽  
Ana E. Rodríguez-Vicente ◽  
Miguel Quijada-Álamo ◽  
José-Ángel Hernández-Rivas ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Gene Mutations ◽  
Lymphocytic Leukemia ◽  
Genetic Biomarkers ◽  
Research Activities ◽  
Routine Clinical Setting ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing ◽  
Over Time

The knowledge of chronic lymphocytic leukemia (CLL) has progressively deepened during the last forty years. Research activities and clinical studies have been remarkably fruitful in novel findings elucidating multiple aspects of the pathogenesis of the disease, improving CLL diagnosis, prognosis and treatment. Whereas the diagnostic criteria for CLL have not substantially changed over time, prognostication has experienced an expansion with the identification of new biological and genetic biomarkers. Thanks to next-generation sequencing (NGS), an unprecedented number of gene mutations were identified with potential prognostic and predictive value in the 2010s, although significant work on their validation is still required before they can be used in a routine clinical setting. In terms of treatment, there has been an impressive explosion of new approaches based on targeted therapies for CLL patients during the last decade. In this current chemotherapy-free era, BCR and BCL2 inhibitors have changed the management of CLL patients and clearly improved their prognosis and quality of life. In this review, we provide an overview of these novel advances, as well as point out questions that should be further addressed to continue improving the outcomes of patients.

scholarly journals Assuring the Quality of Next-Generation Sequencing in Clinical Microbiology and Public Health Laboratories

2016 ◽  
Vol 54 (12) ◽  
pp. 2857-2865 ◽  
Author(s):  
Amy S. Gargis ◽  
Lisa Kalman ◽  
Ira M. Lubin
Keyword(s):  
Public Health ◽  
Next Generation Sequencing ◽  
Clinical Microbiology ◽  
Next Generation ◽  
Standards And Guidelines ◽  
Control Procedures ◽  
Next Generation Sequencing Ngs ◽  
Professional Guidelines ◽  
Generation Sequencing

Clinical microbiology and public health laboratories are beginning to utilize next-generation sequencing (NGS) for a range of applications. This technology has the potential to transform the field by providing approaches that will complement, or even replace, many conventional laboratory tests. While the benefits of NGS are significant, the complexities of these assays require an evolving set of standards to ensure testing quality. Regulatory and accreditation requirements, professional guidelines, and best practices that help ensure the quality of NGS-based tests are emerging. This review highlights currently available standards and guidelines for the implementation of NGS in the clinical and public health laboratory setting, and it includes considerations for NGS test validation, quality control procedures, proficiency testing, and reference materials.

scholarly journals Performance Optimization of a Parallel Error Correction Tool

2021 ◽  
Vol 7 (1) ◽  
pp. 34
Author(s):  
Marco Martínez-Sánchez ◽  
Roberto R. Expósito ◽  
Juan Touriño
Keyword(s):  
Big Data ◽  
Next Generation Sequencing ◽  
Error Correction ◽  
Performance Optimization ◽  
Core Cluster ◽  
Improved Performance ◽  
Next Generation Sequencing Ngs ◽  
Spark Framework ◽  
Generation Sequencing

Due to the continuous development in the field of Next Generation Sequencing (NGS) technologies that have allowed researchers to take advantage of greater genetic samples in less time, it is a matter of relevance to improve the existing algorithms aimed at the enhancement of the quality of those generated reads. In this work, we present a Big Data tool implemented upon the open-source Apache Spark framework that is able to execute validated error-correction algorithms at an improved performance. The experimental evaluation conducted on a multi-core cluster has shown significant improvements in execution times, providing a maximum speedup of 9.5 over existing error correction tools when processing an NGS dataset with 25 million reads.

scholarly journals Optimization of DNA extraction from human urinary samples for mycobiome community profiling

2018 ◽  
Author(s):  
A. Lenore Ackerman ◽  
Jennifer Tash Anger ◽  
Muhammad Umair Khalique ◽  
James E Ackerman ◽  
Jie Tang ◽  
...  
Keyword(s):  
Microbial Community ◽  
Next Generation Sequencing ◽  
Dna Extraction ◽  
Urine Samples ◽  
Next Generation ◽  
Community Profiling ◽  
Optimized Protocol ◽  
Culture Independent ◽  
Community Representation ◽  
Generation Sequencing

Abstract Introduction. Recent data suggest the urinary tract hosts a microbial community of varying composition, even in the absence of infection. Culture-independent methodologies, such as next-generation sequencing of conserved ribosomal DNA sequences, provide an expansive look at these communities, identifying both common commensals and fastidious organisms. A fundamental challenge has been the isolation of DNA representative of the entire resident microbial community, including fungi. Materials and Methods. We evaluated multiple modifications of commonly-used DNA extraction procedures using standardized male and female urine samples, comparing resulting overall, fungal and bacterial DNA yields by quantitative PCR. After identifying protocol modifications that increased DNA yields (lyticase/lysozyme digestion, bead beating, boil/freeze cycles, proteinase K treatment, and carrier DNA use), all modifications were combined for systematic confirmation of optimal protocol conditions. This optimized protocol was tested against commercially available methodologies to compare overall and microbial DNA yields, community representation and diversity by next-generation sequencing (NGS). Results. Overall and fungal-specific DNA yields from standardized urine samples demonstrated that microbial abundances differed significantly among the eight methods used. Methodologies that included multiple disruption steps, including enzymatic, mechanical, and thermal disruption and proteinase digestion, particularly in combination with small volume processing and pooling steps, provided more comprehensive representation of the range of bacterial and fungal species. Concentration of larger volume urine specimens at low speed centrifugation proved highly effective, increasing resulting DNA levels and providing greater microbial representation and diversity. Conclusions. Alterations in the methodology of urine storage, preparation, and DNA processing improve microbial community profiling using culture-independent sequencing methods. Our optimized protocol for DNA extraction from urine samples provided improved fungal community representation. Use of this technique resulted in equivalent representation of the bacterial populations as well, making this a useful technique for the concurrent evaluation of bacterial and fungal populations by NGS.

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