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Thursday, August 27, 2020

Technical Note: Preparing samples for PacBio whole genome sequencing for de novo assembly – Collection and storage

Single Molecule, Real-Time (SMRT) Sequencing uses the natural process of DNA replication to sequence long fragments of native DNA. As such, starting with high-quality, high molecular weight (HMW) genomic DNA (gDNA) will result in better sequencing performance across difficult to sequence regions of the genome. To obtain the highest quality, long DNA it is important to start with sample types compatible with HMW DNA extraction methods. This technical note is intended to give general guidance on sample collection, preparation, and storage across a range of commonly encountered sample types used for SMRT Sequencing whole genome projects. It is important to…

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Thursday, August 27, 2020

Technical Note: Preparing DNA for PacBio HiFi sequencing – Extraction and quality control

Single Molecule, Real-Time (SMRT) Sequencing uses the natural process of DNA replication to sequence long fragments of native DNA in order to produce highly accurate long reads, or HiFi reads. As such, starting with high-quality, high molecular weight (HMW) genomic DNA (gDNA) will result in longer libraries and better performance during sequencing. This technical note is intended to give recommendations, tips and tricks for the extraction of DNA, as well as assessing and preserving the quality and size of your DNA sample to be used for HiFi sequencing.

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Sunday, September 22, 2019

Robust and effective methodologies for cryopreservation and DNA extraction from anaerobic gut fungi.

Cell storage and DNA isolation are essential to developing an expanded suite of microorganisms for biotechnology. However, many features of non-model microbes, such as an anaerobic lifestyle and rigid cell wall, present formidable challenges to creating strain repositories and extracting high quality genomic DNA. Here, we establish accessible, high efficiency, and robust techniques to store lignocellulolytic anaerobic gut fungi long term without specialized equipment. Using glycerol as a cryoprotectant, gut fungal isolates were preserved for a minimum of 23 months at -80 °C. Unlike previously reported approaches, this improved protocol is non-toxic and rapid, with samples surviving twice as long…

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Sunday, September 22, 2019

Protocol: a versatile, inexpensive, high-throughput plant genomic DNA extraction method suitable for genotyping-by-sequencing.

The recent development of next-generation sequencing DNA marker technologies, such as genotyping-by-sequencing (GBS), generates thousands of informative single nucleotide polymorphism markers in almost any species, regardless of genomic resources. This enables poorly resourced or “orphan” crops/species access to high-density, high-throughput marker platforms which have revolutionised population genetics studies and plant breeding. DNA quality underpins success of GBS methods as the DNA must be amenable to restriction enzyme digestion and sequencing. A barrier to implementing GBS technologies is access to inexpensive, high-throughput extraction methods that yield sequencing-quality genomic DNA (gDNA) from plants. Several high-throughput DNA extraction methods are available, but typically…

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Sunday, September 22, 2019

Fast and inexpensive protocols for consistent extraction of high quality DNA and RNA from challenging plant and fungal samples for high-throughput SNP genotyping and sequencing applications.

Modern genotyping techniques, such as SNP analysis and genotyping by sequencing (GBS), are hampered by poor DNA quality and purity, particularly in challenging plant species, rich in secondary metabolites. We therefore investigated the utility of a pre-wash step using a buffered sorbitol solution, prior to DNA extraction using a high salt CTAB extraction protocol, in a high throughput or miniprep setting. This pre-wash appears to remove interfering metabolites, such as polyphenols and polysaccharides, from tissue macerates. We also investigated the adaptability of the sorbitol pre-wash for RNA extraction using a lithium chloride-based protocol. The method was successfully applied to a…

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Sunday, September 22, 2019

Improved nucleic acid extraction protocols for Ganoderma boninense, G. miniatocinctum and G. tornatum.

The first and most crucial step of all molecular techniques is to isolate high quality and intact nucleic acids. However, DNA and RNA isolation from fungal samples are usually difficult due to the cell walls that are relatively unsusceptible to lysis and often resistant to traditional extraction procedures. Although there are many extraction protocols for Ganoderma species, different extraction protocols have been applied to different species to obtain high yields of good quality nucleic acids, especially for genome and transcriptome sequencing. Ganoderma species, mainly G. boninense causes the basal stem rot disease, a devastating disease that plagues the oil palm…

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Friday, July 19, 2019

Extraction of high-molecular-weight genomic DNA for long-read sequencing of single molecules.

De novo sequencing of complex genomes is one of the main challenges for researchers seeking high-quality reference sequences. Many de novo assemblies are based on short reads, producing fragmented genome sequences. Third-generation sequencing, with read lengths >10 kb, will improve the assembly of complex genomes, but these techniques require high-molecular-weight genomic DNA (gDNA), and gDNA extraction protocols used for obtaining smaller fragments for short-read sequencing are not suitable for this purpose. Methods of preparing gDNA for bacterial artificial chromosome (BAC) libraries could be adapted, but these approaches are time-consuming, and commercial kits for these methods are expensive. Here, we present…

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Sunday, July 7, 2019

Whole-genome sequencing for comparative genomics and de novo genome assembly.

Next-generation sequencing technologies for whole-genome sequencing of mycobacteria are rapidly becoming an attractive alternative to more traditional sequencing methods. In particular this technology is proving useful for genome-wide identification of mutations in mycobacteria (comparative genomics) as well as for de novo assembly of whole genomes. Next-generation sequencing however generates a vast quantity of data that can only be transformed into a usable and comprehensible form using bioinformatics. Here we describe the methodology one would use to prepare libraries for whole-genome sequencing, and the basic bioinformatics to identify mutations in a genome following Illumina HiSeq or MiSeq sequencing, as well as…

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Sunday, July 7, 2019

Modified low-salt CTAB extraction of high-quality DNA from contaminant-rich tissues.

The increasing use of high-throughput sequencing platforms has made the isolation of pure, high molecular weight DNA a primary concern for studies of a diverse range of organisms. Purification of DNA remains a significant challenge in many tissue and sample types due to various organic and inorganic molecules that coprecipitate with nucleic acids. Molluscs, for example, contain high concentrations of polysaccharides which often coprecipitate with DNA and can inhibit downstream enzymatic reactions. We modified a low-salt CTAB (MoLSC) extraction protocol to accommodate contaminant-rich animal tissues and compared this method to a standard CTAB extraction protocol and two commercially available animal…

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Sunday, July 7, 2019

Extraction of high molecular weight DNA from fungal rust spores for long read sequencing.

Wheat rust fungi are complex organisms with a complete life cycle that involves two different host plants and five different spore types. During the asexual infection cycle on wheat, rusts produce massive amounts of dikaryotic urediniospores. These spores are dikaryotic (two nuclei) with each nucleus containing one haploid genome. This dikaryotic state is likely to contribute to their evolutionary success, making them some of the major wheat pathogens globally. Despite this, most published wheat rust genomes are highly fragmented and contain very little haplotype-specific sequence information. Current long-read sequencing technologies hold great promise to provide more contiguous and haplotype-phased genome…

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Sunday, July 7, 2019

A simple thermoplastic substrate containing hierarchical silica lamellae for high-molecular-weight DNA extraction.

An inexpensive, magnetic thermoplastic nanomaterial is developed utilizing a hierarchical layering of micro- and nanoscale silica lamellae to create a high-surface-area and low-shear substrate capable of capturing vast amounts of ultrahigh-molecular-weight DNA. Extraction is performed via a simple 45 min process and is capable of achieving binding capacities up to 1 000 000 times greater than silica microparticles. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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Sunday, July 7, 2019

DNA extraction protocols for whole-genome sequencing in marine organisms.

The marine environment harbors a large proportion of the total biodiversity on this planet, including the majority of the earths’ different phyla and classes. Studying the genomes of marine organisms can bring interesting insights into genome evolution. Today, almost all marine organismal groups are understudied with respect to their genomes. One potential reason is that extraction of high-quality DNA in sufficient amounts is challenging for many marine species. This is due to high polysaccharide content, polyphenols and other secondary metabolites that will inhibit downstream DNA library preparations. Consequently, protocols developed for vertebrates and plants do not always perform well for…

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Sunday, July 7, 2019

Small- and Large-Scale High Molecular Weight Genomic DNA Extraction from Planarians.

High-quality genomic DNA extraction is a starting point for many downstream applications in modern molecular biology. Here, we describe a simple method for isolating high molecular weight genomic DNA from planarians. The method is based on tissue lysis by a mixture of a chaotropic salt and detergent followed by organic extraction to remove proteins and lipids followed by a postpurification step to remove contaminating polysaccharides. The isolated DNA is of high molecular weight and compatible with polymerase chain reaction, cloning, or next-generation sequencing library preparation.

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