Query: DNA extraction   ont

title: Plant DNA extraction and preparation for ONT sequencing v1

title: DNA extraction of microbial DNA directly from infected tissue: an optimized protocol for use in nanopore sequencing
abstract: Identification of bacteria causing tissue infections can be comprehensive and, in the cases of non or slow growing bacteria, near impossible with conventional methods. Performing shotgun metagenomic sequencing on bacterial DNA extracted directly from the infected tissue may improve time to diagnosis and targeted treatment considerably. However, infected tissue consists mainly of human DNA (hDNA) which hampers bacterial identification. In this proof of concept study, we present a modified version of the Ultra Deep Microbiome Prep kit for DNA extraction procedure, removing additional human DNA. Tissue biopsies from 3 patients with orthopedic implant related infections containing varying degrees of Staphylococcus aureus were included. Subsequent DNA shotgun metagenomic sequencing using Oxford Nanopore Technologies' (ONT) MinION platform and ONTs EPI2ME WIMP and ARMA bioinformatic workflows for microbe and antibiotic resistance genes identification, respectively. The modified DNA extraction protocol led to an additional ~10 fold reduction of human DNA while preserving S. aureus DNA. Including the DNA sequencing and bioinformatics analyses, the presented protocol has the potential of identifying the infection causing pathogen in infected tissue within 7 hours after biopsy. However, due to low number of S. aureus reads, positive identification of antibiotic resistance genes was not possible.

title: DNA extraction from gram negative bacteria for ONT MinION sequencing

title: Evaluation of DNA extraction methods for the detection of Cytomegalovirus in dried blood spots.
abstract: Dried blood spots (DBS) are collected universally from newborns and may be valuable for the diagnosis of congenital Cytomegalovirus (CMV) infection. The reported analytical sensitivity for DBS testing compared to urine or saliva varies greatly across CMV studies. The purpose of this study was to directly compare the performance of various DNA extraction methods for identification of CMV in DBS including those used most often in CMV studies.  Grade 903 filter paper cards were spotted with blood samples from 25 organ transplant recipients who had confirmed CMV viremia. Six DNA extraction methods were compared for relative yield of viral and cellular DNA: 2 manual solution based methods (Gentra Puregene, thermal shock) 2 manual silica column based methods (QIAamp DNA Mini, QIAamp DNA Investigator) and 2 automated methods (M48 MagAttract Mini, QIAcube Investigator) DBS extractions were performed in triplicate followed by real time quantitative PCR (qPCR)  For extraction of both viral and cellular DNA, two methods (QIAamp DNA Investigator and thermal shock) consistently gave the highest yields, and two methods (M48 MagAttract Mini and QIAamp DNA Mini) consistently gave the lowest yields. There was an average 3 fold difference in DNA yield between the highest and lowest yield methods.  The choice of DNA extraction method is a major factor in the ability to detect low levels of CMV in DBS and can largely account for the wide range of DBS sensitivities reported in studies to date.

title: Choice of bacterial DNA extraction method from fecal material influences community structure as evaluated by metagenomic analysis
abstract: BackgroundIn recent years, studies on the human intestinal microbiota have attracted tremendous attention. Application of next generation sequencing for mapping of bacterial phylogeny and function has opened new doors to this field of research. However, little attention has been given to the effects of choice of methodology on the output resulting from such studies.ResultsIn this study we conducted a systematic comparison of the DNA extraction methods used by the two major collaborative efforts: The European MetaHIT and the American Human Microbiome Project (HMP) Additionally, effects of homogenizing the samples before extraction were addressed. We observed significant differences in distribution of bacterial taxa depending on the method. While eukaryotic DNA was most efficiently extracted by the MetaHIT protocol, DNA from bacteria within the Bacteroidetes phylum was most efficiently extracted by the HMP protocol.ConclusionsWhereas it is comforting that the inter individual variation clearly exceeded the variation resulting from choice of extraction method, our data highlight the challenge of comparing data across studies applying different methodologies.

title: Pathogen Quantitation in Complex Matrices: A Multi Operator Comparison of DNA Extraction Methods with a Novel Assessment of PCR Inhibition
abstract: Mycobacterium bovis is the aetiological agent of bovine tuberculosis (bTB) an important recrudescent zoonosis, significantly increasing in British herds in recent years. Wildlife reservoirs have been identified for this disease but the mode of transmission to cattle remains unclear. There is evidence that viable M. bovis cells can survive in soil and faeces for over a year.  We report a multi operator blinded trial for a rigorous comparison of five DNA extraction methods from a variety of soil and faecal samples to assess recovery of M. bovis via real time PCR detection. The methods included four commercial kits: the QIAamp Stool Mini kit with a pre treatment step, the FastDNA(r) Spin kit, the UltraClean(tm) and PowerSoil(tm) soil kits and a published manual method based on phenol:chloroform purification, termed Griffiths. M. bovis BCG Pasteur spiked samples were extracted by four operators and evaluated using a specific real time PCR assay. A novel inhibition control assay was used alongside spectrophotometric ratios to monitor the level of inhibitory compounds affecting PCR, DNA yield, and purity. There were statistically significant differences in M. bovis detection between methods of extraction and types of environmental samples; no significant differences were observed between operators. Processing times and costs were also evaluated. To improve M. bovis detection further, the two best performing methods, FastDNA(r) Spin kit and Griffiths, were optimised and the ABI TaqMan environmental PCR Master mix was adopted, leading to improved sensitivities.  M. bovis was successfully detected in all environmental samples; DNA extraction using FastDNA(r) Spin kit was the most sensitive method with highest recoveries from all soil types tested. For troublesome faecal samples, we have used and recommend an improved assay based on a reduced volume, resulting in detection limits of 4.25x105 cells g 1 using Griffiths and 4.25x106 cells g 1 using FastDNA(r) Spin kit.

title: Metagenomic comparison of direct and indirect soil DNA extraction approaches.
abstract: Full pyrosequencing runs of both direct extracted (high yield, low DNA size) and indirect extracted DNA (low yield, high DNA size) from the same prairie soil show that the sequence distribution of the majority of the metabolic functions and species detected were statistically similar. Although some microbial functions differed at the 95% confidence interval in bootstrap analyses, the overall functional diversity was the same.

title: Techniques of dental DNA extraction: Some operative experiences.
abstract: This study presents and discusses the possibility of multiple dental DNA extraction at different times using an endodontic technique. We chose a mixed sample of twenty teeth (decayed, filled, different kinds of teeth, etc. and performed two different accesses to the pulp cavity to collect samples of dental tissue useful for DNA extraction. The Identifiler((r) and Minifiler((r) kits were used respectively for the first and second genetic analysis. The research's most relevant findings are the possibility of successfully repeating dental DNA extractions, and that a dental element can be considered as a DNA source that can be reused even after prolonged time elapses. The study's results document a simple and efficient methodology for dental identification based on the choice of dental elements, endodontic techniques and the use of the Minifiler((r) kit for genetic analysis.

title: Buccal cells DNA extraction to obtain high quality human genomic DNA suitable for polymorphism genotyping by PCR RFLP and Real Time PCR
abstract: The aim of this study was to evaluate, by PCR RFLP and Real time PCR, the yield and quality of genomic DNA collected from buccal cells by mouthwash after different storage times at room temperature.  A group of volunteers was recruited to collect buccal cells using a mouthwash solution. The collected solution was divided into 3 tubes, one tube were used for immediate extraction and the remaining received ethanol and were kept at room temperature for 4 and 8 days followed by DNA extraction. The concentration, purity and integrity of the DNA were determined using spectrophotometry and electrophoresis. DNA quality differences among the three incubation times were also evaluated for genotyping EGF +61 A/G (rs 4444903) polymorphism by PCR RFLP and for IRF6 polymorphism (rs 17015215) using Real Time PCR.  There was no significant difference of DNA yield (p=0.75) and purity (p=0.86) among the three different incubation times. DNA obtained from different incubation times presented high molecular weight. The PCR RFLP and Real time PCR reactions were successfully performed for all DNA samples, even those extracted after 8 days of incubation. All samples genotyped by Real Time PCR presented C allele for IRF6 gene polymorphism (homozygous: CC; heterozygous: CT) and the C allele was used as a reference for Ct values. The samples presented the same genotype for the different times in both techniques.  We demonstrated that the method described herein is simple and low cost, and that DNA can be extracted and PCR amplified after storage in mouthwash solution at room temperature.

title: A rapid genotyping method for an obligate fungal pathogen, Puccinia striiformis f.sp. tritici, based on DNA extraction from infected leaf and Multiplex PCR genotyping
abstract: BackgroundPuccinia striiformis f.sp. tritici (PST) an obligate fungal pathogen causing wheat yellow/stripe rust, a serious disease, has been used to understand the evolution of crop pathogen using molecular markers. However, numerous questions regarding its evolutionary history and recent migration routes still remains to be addressed, which need the genotyping of a large number of isolates, a process that is limited by both DNA extraction and genotyping methods. To address the two issues, we developed here a method for direct DNA extraction from infected leaves combined with optimized SSR multiplexing.FindingsWe report here an efficient protocol for direct fungal DNA extraction from infected leaves, avoiding the costly and time consuming step of spore multiplication. The genotyping strategy we propose, amplified a total of 20 SSRs in three Multiplex PCR reactions, which were highly polymorphic and were able to differentiate different PST populations with high efficiency and accuracy.ConclusionThese two developments enabled a genotyping strategy that could contribute to the development of molecular epidemiology of yellow rust disease, both at a regional or worldwide scale.