Method For Preparation Of DNA Research Articles

One-pot method for preparing DNA, RNA, and protein for multiomics analysis

Typical multiomics studies employ separate methods for DNA, RNA, and protein sample preparation, which is labor intensive, costly, and prone to sampling bias. We describe a method for preparing high-quality, sequencing-ready DNA and RNA, and either intact proteins or mass-spectrometry-ready peptides for whole proteome analysis from a single sample. This method utilizes a reversible protein tagging scheme to covalently link all proteins in a lysate to a bead-based matrix and nucleic acid precipitation and selective solubilization to yield separate pools of protein and nucleic acids. We demonstrate the utility of this method to compare the genomes, transcriptomes, and proteomes of four triple-negative breast cancer cell lines with different degrees of malignancy. These data show the involvement of both RNA and associated proteins, and protein-only dependent pathways that distinguish these cell lines. We also demonstrate the utility of this multiomics workflow for tissue analysis using mouse brain, liver, and lung tissue.

Improved DNA Extraction and Amplification Strategy for 16S rRNA Gene Amplicon-Based Microbiome Studies.

Next-generation sequencing technology has driven the rapid advancement of human microbiome studies by enabling community-level sequence profiling of microbiomes. Although all microbiome sequencing methods depend on recovering the DNA from a sample as a first critical step, lysis methods can be a major determinant of microbiome profile bias. Gentle enzyme-based DNA preparation methods preserve DNA quality but can bias the results by failing to open difficult-to-lyse bacteria. Mechanical methods like bead beating can also bias DNA recovery because the mechanical energy required to break tougher cell walls may shear the DNA of the more easily lysed microbes, and shearing can vary depending on the time and intensity of beating, influencing reproducibility. We introduce a non-mechanical, non-enzymatic, novel rapid microbial DNA extraction procedure suitable for 16S rRNA gene-based microbiome profiling applications that eliminates bead beating. The simultaneous application of alkaline, heat, and detergent ('Rapid' protocol) to milligram quantity samples provided consistent representation across the population of difficult and easily lysed bacteria equal to or better than existing protocols, producing sufficient high-quality DNA for full-length 16S rRNA gene PCR. The novel 'Rapid' method was evaluated using mock bacterial communities containing both difficult and easily lysed bacteria. Human fecal sample testing compared the novel Rapid method with a standard Human Microbiome Project (HMP) protocol for samples from lung cancer patients and controls. DNA recovered from both methods was analyzed using 16S rRNA gene sequencing of the V1V3 and V4 regions on the Illumina platform and the V1V9 region on the PacBio platform. Our findings indicate that the 'Rapid' protocol consistently yielded higher levels of Firmicutes species, which reflected the profile of the bacterial community structure more accurately, which was confirmed by mock community evaluation. The novel 'Rapid' DNA lysis protocol reduces population bias common to bead beating and enzymatic lysis methods, presenting opportunities for improved microbial community profiling, combined with the reduction in sample input to 10 milligrams or less, and it enables rapid transfer and simultaneous lysis of 96 samples in a standard plate format. This results in a 20-fold reduction in sample handling time and an overall 2-fold time advantage when compared to widely used commercial methods. We conclude that the novel 'Rapid' DNA extraction protocol offers a reliable alternative for preparing fecal specimens for 16S rRNA gene amplicon sequencing.

Detection of Esca‐associated fungi in grapevine trunks using loop‐mediated isothermal amplification (LAMP) assays

AbstractEsca is a grapevine trunk disease (GTD) that is caused by filamentous fungi. It is responsible for considerable economic losses in viniculture on a global scale. Despite many unknown factors contributing to the development of symptoms in affected plants, Phaeoacremonium minimum (PMI), Phaeomoniella chlamydospora (PCH) and Fomitiporia mediterranea (FMED) are generally considered as the main causative fungal species. Early detection and specific identification of these pathogens therefore play an important role in disease control and evaluation of suitable countermeasures. In this study, loop‐mediated isothermal amplification (LAMP) assays were developed for each of the three pathogens. A genome‐based approach was applied for detection and selection of unique target DNA sequences. The designed primer sets showed overall good specificities, with some observed cross‐reactions towards closely related Phaeoacremonium species for the PMI primer set. The developed assays had detection limits of 100 pg (FMED, PMI) and 1 pg (PCH) per reaction (corresponding to 1460 [FMED]; 1950 [PMI]; 342 [PCH] genome copies per reaction). The application of the assays to field samples was demonstrated by testing individual infected grapevine trunks from two European viticultural regions using crude DNA obtained in a rapid sample preparation step. LAMP assay results matched those of PCR following a conventional DNA extraction protocol. The study showed that LAMP‐based rapid molecular detection of major Esca agents can serve as a useful tool for further research and surveillance of a highly devastating grapevine disease. The application of computer‐based whole genome comparison between target and non‐target species for the identification of unique target sequences as the basis for LAMP (or PCR) primer design was demonstrated to be a useful approach in species for which scarce sequence information is available. Moreover, the developed method for rapid DNA preparation from grapevine trunks may potentially be adapted to the DNA‐based detection also of other fungal species that cause grapevine trunk diseases.

A Simplified, Efficient and Rapid DNA Extraction Protocol from Rice Grains and Leaf

DNA extraction is very complicated with different plant species because the presence of secondary metabolites that hinder with DNA isolation processes and application like DNA restriction, Gene amplification, as well as gene cloning. A simple method for preparation of rice genomic DNA was developed. The current study expressed comparatively rapid, cost-effective, less time consuming, methods for DNA extraction from seed, young leaves and old leaves of rice genotypes without using liquid nitrogen, phenol, and β–mercaptoethanol in extraction buffer. Using this method, high quality and quantity of genomic DNA was get from 0.5 g of rice seed, young leaves and 75 days old leavesand extracted total nucleic acids (genomic DNA) were amplified by employing single sequence repeats (SSR) or microsatellite markers, which produced reproducible results.This protocol resolves the problems of DNA degradation, contamination, and low yield due to binding and/or coprecipitation with starches and polysaccharides.

Selection of Antibiotic-Resistant Bacterial Strains and Identification of Genomic Alterations by Whole-Genome Sequencing: Using Staphylococcus aureus and Oxacillin Resistance as an Example.

Here, we discuss methods for the selection of antibiotic-resistant bacteria and the use of high-throughput whole-genome sequencing for the identification of the underlying mutations. We comment on sample requirements and the choice of specific DNA preparation methods depending on the strain used and briefly introduce a workflow we use for the selection of Staphylococcus aureus strains with increased oxacillin resistance and identification of genomic alterations.

Rapid preparation of Candida genomic DNA: combined use of enzymatic digestion and thermal disruption

Nucleic acid based molecular technologies are the most promising tools for the early diagnosis of Candida infection. A simple and effective DNA preparation method is of critical for standardizing and applying molecular diagnostics in clinic laboratories. The goal of this study was to develop a Candida DNA preparation method that was quick to do, easy to perform, and bio-safe. Snailase and lyticase were screened and combined in this work to enhance the lysis of Candida cells. The lysis solution composition and metal bath were optimized to boost amplification efficiency and biosafety. A duplex real-time PCR was established to evaluate the sensitivity and specificity of the preparation method. Using the supernatant from the rapid preparation method as templates, the duplex PCR sensitivities for five common Candida species were determined to be as low as 100 CFUs. When compared to conventional preparation methods, the samples prepared by our method showed higher PCR detection sensitivity. PCR identification and ITS sequencing were 100% consistent, which was better than biochemical identification. This study demonstrates a rapid method for Candida DNA preparation that has the potential to be used in clinical laboratories. Meanwhile, the practical application of the method for clinical samples needs to be proven in future investigations.

Cost-Effective PCR-Based Identification of Tunga penetrans (Siphonaptera) Larvae Extracted from Soil Samples Containing PCR Inhibitor-Rich Material

Tungiasis is a neglected tropical disease caused by skin-penetrating female Tunga penetrans fleas. Although tungiasis causes severe health problems, its ecology is poorly understood and morphological descriptions of the larvae are unavailable. To identify T. penetrans immature stages and sites where they develop, diagnostic PCRs are required. However, flea larvae feed on soil organic matter rich in PCR inhibitors. Here, three DNA preparation methods, including a soil DNA kit that removes inhibitors, a simple ammonium acetate precipitation approach (AmAcet) and a crude lysate of larvae (CL), were combined with amplification by the highly processive FIREPol® Taq or the inhibitor-resistant Phusion® polymerase. Independent of the polymerase used, the frequency of successful amplification, Cq values and PCR efficacies for the low-cost CL and AmAcet methods were superior to the commercial kit for amplification of a 278 bp partial internal transcribed spacer-2 (ITS-2) and a 730 bp pan-Siphonaptera cytochrome oxidase II PCR. For the CL method combined with Phusion® polymerase, the costs were approximately 20-fold lower than for the methods based on the soil DNA kit, which is a considerable advantage in resource-poor settings. The ITS-2 PCR did not amplify Ctenocephalides felis genomic or Tunga trimammilata ITS-2 plasmid DNA, meaning it can be used to specifically identify T. penetrans.

A Rapid and Simple Method for DNA Preparation of Magnaporthe oryzae from Single Rice Blast Lesions for PCR-Based Molecular Analysis.

Rice blast is one of the most destructive diseases of rice worldwide, and the causative agent is the filamentous ascomycete Magnaporthe oryzae. With the successful cloning of more and more avirulence genes from M. oryzae, the direct extraction of M. oryzae genomic DNA from infected rice tissue would be useful alternative for rapid monitoring of changes of avirulence genes without isolation and cultivation of the pathogen. In this study, a fast, low-cost and reliable method for DNA preparation of M. oryzae from a small piece of infected single rice leaf or neck lesion was established. This single step method only required 10 min for DNA preparation and conventional chemical reagents commonly found in the laboratory. The AvrPik and AvrPi9 genes were successfully amplified with the prepared DNA. The expected DNA fragments from 570 bp to 1,139 bp could be amplified even three months after DNA preparation. This method was also suitable for DNA preparation from M. oryzae strains stored on the filter paper. All together these results indicate that the DNA preparation method established in this study is reliable, and could meet the basic needs for polymerase chain reaction-based analysis of M. oryzae.

GMEmbeddings: An R Package to Apply Embedding Techniques to Microbiome Data.

Large-scale microbiome studies investigating disease-inducing microbial roles base their findings on differences between microbial count data in contrasting environments (e.g., stool samples between cases and controls). These microbiome survey studies are often impeded by small sample sizes and database bias. Combining data from multiple survey studies often results in obvious batch effects, even when DNA preparation and sequencing methods are identical. Relatedly, predictive models trained on one microbial DNA dataset often do not generalize to outside datasets. In this study, we address these limitations by applying word embedding algorithms (GloVe) and PCA transformation to ASV data from the American Gut Project and generating translation matrices that can be applied to any 16S rRNA V4 region gut microbiome sequencing study. Because these approaches contextualize microbial occurrences in a larger dataset while reducing dimensionality of the feature space, they can improve generalization of predictive models that predict host phenotype from stool associated gut microbiota. The GMEmbeddings R package contains GloVe and PCA embedding transformation matrices at 50, 100 and 250 dimensions, each learned using ∼15,000 samples from the American Gut Project. It currently supports the alignment, matching, and matrix multiplication to allow users to transform their V4 16S rRNA data into these embedding spaces. We show how to correlate the properties in the new embedding space to KEGG functional pathways for biological interpretation of results. Lastly, we provide benchmarking on six gut microbiome datasets describing three phenotypes to demonstrate the ability of embedding-based microbiome classifiers to generalize to independent datasets. Future iterations of GMEmbeddings will include embedding transformation matrices for other biological systems. Available at: https://github.com/MaudeDavidLab/GMEmbeddings.

High-Throughput and Low-Cost Genotyping Method for Plant Genome Editing.

Genome editing technologies have revolutionized genetic studies in the life sciences community in recent years. The application of these technologies allows researchers to conveniently generate mutations in almost any gene of interest. This is very useful for species such as maize that have complex genomes and lack comprehensive mutant collections. With the improvement of genome editing tools and transformation methods, these technologies are also widely used to assist breeding research and implementation in maize. However, the detection and genotyping of genomic edits rely on low-throughput, high-cost methods, such as traditional agarose gel electrophoresis and Sanger sequencing. This article describes a method to barcode the target regions of genomic edits from many individuals by low-cost polymerase chain reaction (PCR) amplification. It also employs next-generation sequencing (NGS) to genotype the genome-edited plants at high throughput and low cost. This protocol can be used for initial screening of genomic edits as well as derived population genotyping on a small or large scale, at high efficiency and low cost. © 2021 Wiley Periodicals LLC. Basic Protocol 1: A fast genomic DNA preparation method from genome edited plants Basic Protocol 2: Barcoding the amplicons of edited regions from each individual by two rounds of PCR Basic Protocol 3: Bioinformatics analysis.

PCR-detectable Candida DNA exists a short period in the blood of systemic candidiasis murine model.

Invasive candidiasis is a major challenge to clinical medicine today. However, traditional fungal diagnostic techniques and empirical treatments have shown great limitations. Although efforts are necessarily needed in methodology standardization and multicenter validation, polymerase chain reaction (PCR) is a very promising assay in detecting fungal pathogens. Using a “heat-shock” DNA preparation method, a rapid and simple PCR protocol for quantification of the Candida albicans (C. albicans) ribosomal DNA was established. The PCR assay could detect Candida DNA as low as 10 CFU/mL in samples prepared by the heat-shock protocol, without any cross-reaction with DNA prepared from other Candida spp. and bacterial pathogens. For simulated blood samples, the PCR test sensitivity of whole blood samples was better than that of plasma and blood cells. In the systemic candidiasis murine model, detectable DNA was only observed within 24 h after C. albicans SC5314 injection, which is much shorter than that observed in the kidney.

A Simple and Cost-Effective DNA Preparation Method Suitable for High-Throughput PCR Quantification of Hepatitis B Virus Genomes.

Hepatitis B virus (HBV) is a para-retrovirus that reverse transcribes its pregenomic RNA into relaxed circular DNA inside viral nucleocapsids. The number of HBV genomes produced in vitro is typically quantified using commercial silica-membrane-based nucleic acid purification kits to isolate total DNA followed by HBV-specific quantitative PCR (qPCR). However, despite the convenience of commercial kits, this procedure is costly and time-consuming due to multiple centrifugation steps, which produce unnecessary waste. Here, we report a rapid, cost-effective, and environmentally friendly total DNA preparation method. The assay is based on the simple incubation of detergent and proteinase K with cells or cell-free supernatants to permeabilize cells and disrupt viral particles. After heat inactivation and subsequent centrifugation to clear the lysates, DNA samples are directly subjected to qPCR to quantify HBV genomes. As a proof of concept, the assay was developed in 12-well plates to assess intra- and extracellular HBV genome equivalents (GEqs) of stably viral-replicating cell lines (e.g., HepAD38) and HBV-infected HepG2-NTCP cells, both treated with lamivudine (LMV), an HBV replication inhibitor. Viral DNA was also prepared from the serum of patients chronically infected with HBV. To validate the assay, a representative commercial DNA isolation kit was used side-by-side to isolate intra- and extracellular HBV DNA. Both methods yielded comparable amounts of HBV GEqs with comparable LMV 50% efficient concentration (EC50) values. The assay was subsequently adapted to 96- and 384-well microtiter plates using HepAD38 cells. The EC50 values were comparable to those obtained in 12-well plates. In addition, the calculated coefficient of variation, Z’ values, and assay window demonstrated high reproducibility and quality. We devised a novel, robust, reproducible, high-throughput microtiter plate DNA preparation method suitable for quantifying HBV GEqs by qPCR analysis. This strategy enables rapid and convenient quantitative analysis of multiple viral DNA samples in parallel to investigate intracellular HBV replication and the secretion of DNA-containing viral particles.

Introduction of a bead beating step improves fungal DNA extraction from selected patient specimens

In immunocompromised patients a colonisation with fungi carries the risk to develop serious invasive fungal infection. An early detection is therefore important, but not optimal hitherto. Fortunately, molecular genetic methods have increased the sensitivity of fungal detection and limited the time, until results are available. However, their success depends on an efficient extraction of genomic DNA from the fungal cell in the given diagnostic specimen.To improve the routine DNA preparation method for yeasts and moulds, the impact of bead beating on fungal DNA release was evaluated. PBS, blood and respiratory rinse were spiked with Candida glabrata or Aspergillus fumigatus. DNA was extracted by mechanical bead beating in addition to the different steps of the DNA preparation protocol, which comprised liquid nitrogen treatment, proteinase K digestion and DNA isolation using the EZ1 DNA Tissue Kit and Workstation. In every method variant tested, treatment with liquid nitrogen did not improve the DNA release. Bead beating once followed by proteinase K digestion and EZ1-work-up led to the highest DNA release from fungus, spiked in PBS, and increased the extracted DNA amount of C. glabrata about 100-fold and of A. fumigatus about 10-fold in relation to sole EZ1-work-up. In fungus-spiked respiratory rinse and blood, highest increase in DNA release was measured after triple bead beating with simultaneous proteinase K digestion. Fungal DNA release of C. glabrata increased for >100-fold in respiratory rinse and for >1000-fold in blood and of A. fumigatus for >10-fold in respiratory rinse and about 5- to 10-fold in blood.The data of this study clearly demonstrate that preparation of fungal DNA from human specimens is optimized by introduction of a bead beating step to the conventional DNA-preparation method without the necessity of a liquid nitrogen step.

Draft Genome Sequence of Bacillus velezensis BZR 336g, a Plant Growth-Promoting Antifungal Biocontrol Agent Isolated from Winter Wheat.

Bacillus velezensis strain BZR 336g is a plant growth-promoting rhizobacterium isolated from a winter wheat rhizoplane from the Krasnodar region in Russia. In this study, we report the genome, including genes with known phenotypic function, i.e., the biosynthesis of secondary metabolites with fungicidal and plant growth-promoting activities. We sequenced and analyzed the complete BZR 336g genome using two different DNA preparation methods to help us better understand the origin of the antimicrobial and antifungal abilities and to weigh the biocontrol properties of this strain.

Shotgun-Metagenomics on Positive Blood Culture Bottles Inoculated With Prosthetic Joint Tissue: A Proof of Concept Study.

Clinical metagenomics is actively moving from research to clinical laboratories. It has the potential to change the microbial diagnosis of infectious diseases, especially when detection and identification of pathogens can be challenging, such as in prosthetic joint infection (PJI). The application of metagenomic sequencing to periprosthetic joint tissue (PJT) specimens is often challenged by low bacterial load in addition to high level of inhibitor and contaminant host DNA, limiting pathogen recovery. Shotgun-metagenomics (SMg) performed directly on positive blood culture bottles (BCBs) inoculated with PJT may be a convenient approach to overcome these obstacles. The aim was to test if it is possible to perform SMg on PJT inoculated into BCBs for pathogen identification in PJI diagnosis. Our study was conducted as a laboratory method development. For this purpose, spiked samples (positive controls), negative control and clinical tissue samples (positive BCBs) were included to get a comprehensive overview. We developed a method for preparation of bacterial DNA directly from PJT inoculated in BCBs. Samples were processed using MolYsis5 kit for removal of human DNA and DNA extracted with BiOstic kit. High DNA quantity/quality was obtained, and no inhibition was observed during the library preparation, allowing further sequencing process. DNA sequencing reads obtained from the BCBs, presented a low proportion of human reads (<1%) improving the sensitivity of bacterial detection. We detected a 19-fold increase in the number of reads mapping to human in a sample untreated with MolYsis5. Taxonomic classification of clinical samples identified a median of 96.08% (IQR, 93.85–97.07%; range 85.7–98.6%) bacterial reads. Shotgun-metagenomics results were consistent with the results from a conventional BCB culture method, validating our approach. Overall, we demonstrated a proof of concept that it is possible to perform SMg directly on BCBs inoculated with PJT, with potential of pathogen identification in PJI diagnosis. We consider this a first step in research efforts needed to face the challenges presented in PJI diagnoses.

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology.

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly sensitive. This protocol obtains precise measurements of gene expression from single worms or from bulk samples. The protocol presented here provides a novel adaptation of existing methods for complementary DNA (cDNA) preparation coupled to a nanofluidic RT-qPCR platform. The first part of this protocol, named 'Worm-to-CT', allows cDNA production directly from nematodes without the need for prior mRNA isolation. It increases experimental throughput by allowing the preparation of cDNA from 96 worms in 3.5 h. The second part of the protocol uses existing nanofluidic technology to run high-throughput RT-qPCR on the cDNA. This paper evaluates two different nanofluidic chips: the first runs 96 samples and 96 targets, resulting in 9,216 reactions in approximately 1.5 days of benchwork. The second chip type consists of six 12 x 12 arrays, resulting in 864 reactions. Here, the Worm-to-CT method is demonstrated by quantifying mRNA levels of genes encoding heat shock proteins from single worms and from bulk samples. Provided is an extensive list of primers designed to amplify processed RNA for the majority of coding genes within the C. elegans genome.

Simple lysis of bacterial cells for DNA-based diagnostics using hydrophilic ionic liquids

The extraction of nucleic acids from microorganisms for subsequent molecular diagnostic applications is still a tedious and time-consuming procedure. We developed a method for the rapid preparation of genomic DNA from bacteria based on hydrophilic ionic liquids (ILs). First, we tested eight ILs in different buffer systems for their inhibitory effects on quantitative PCR. The cell lysis potential of different IL/buffer combinations was assessed by application on Enterococcus faecalis as a model organism for Gram-positive bacteria. The two best ILs, choline hexanoate and 1-ethyl-3-methylimidazolium acetate, were compared with the reference enzymatic method and two commercial DNA extraction kits. All methods were evaluated on four Gram-positive and four Gram-negative bacterial species that are highly relevant for environmental, food, or clinical diagnostics. In comparison to the reference method, extraction yields of the IL-based procedure were within one order of magnitude for most of the strains. The final protocol for DNA extraction using the two ILs is very low-cost, avoids the use of hazardous chemicals and can be performed in five minutes on a simple heating block. This makes the method ideal for high sample throughput and offers the opportunity for DNA extraction from bacteria in resource-limited settings or even in the field.

Efficient coralline algal psbA mini barcoding and High Resolution Melt (HRM) analysis using a simple custom DNA preparation

Coralline algae form extensive maerl and rhodolith habitats that support a rich biodiversity. Calcium carbonate harvesting as well as trawling activities threatens this ecosystem. Eleven species were recorded so far as maerl-forming in NE Atlantic, but identification based on morphological characters is unreliable. As for most red algae, we now use molecular characters to resolve identification of these taxa. However, obtaining DNA sequences requires time and resource demanding methods. The purpose of our study was to improve methods for achieving simple DNA extraction, amplification, sequencing and sequence analysis to allow robust identification of maerl species and other coralline algae. Our novel and easy DNA preparation method for coralline algae was of sufficient quality for qPCR amplification and sequencing of all 47 tested samples. The new psbA qPCR assay successfully amplified a 350 bp fragment identifying six species and uncovering two new Operational Taxonomic Units. Molecular results were corroborated with anatomical examination using i.e. scanning electron microscopy. Finally, the qPCR assay was coupled with High Resolution Melt analysis that successfully differentiated the closely related species Lithothamnion erinaceum and L. cf. glaciale. This DNA preparation and qPCR technique should vitalize coralline research by reducing time and cost associated with molecular systematics.

A Comparative Analysis of Microbial DNA Preparation Methods for Use With Massive and Branching Coral Growth Forms.

In the last two decades, over 100 studies have investigated the structure of the coral microbiome. However, as yet there are no standardized methods applied to sample preservation and preparation, with different studies using distinct methods. There have also been several comparisons made of microbiome data generated across different studies, which have not addressed the influence of the methodology employed over each of the microbiome datasets. Here, we assess three different preservation methods; salt saturated dimethyl sulfoxide (DMSO) – EDTA, snap freezing with liquid nitrogen and 4% paraformaldehyde solution, and two different preparation methodologies; bead beating and crushing, that have been applied to study the coral microbiome. We compare the resultant bacterial assemblage data for two coral growth forms, the massive coral Goniastrea edwardsi and the branching coral Isopora palifera. We show that microbiome datasets generated from differing preservation and processing protocols are comparable in composition (presence/absence). Significant discrepancies between preservation and homogenization methods are observed in structure (relative abundance), and in the occurrence and dominance of taxa, with rare (low abundance and low occurrence) phylotypes being the most variable fraction of the microbial community. Finally, we provide evidence to support chemical preservation with DMSO as effective as snap freezing samples for generating reliable and robust microbiome datasets. In conclusion, we recommend where possible a standardized preservation and extraction method be taken up by the field to provide the best possible practices for detailed assessments of symbiotic and conserved bacterial associations.

Gel-seq: A Method for Simultaneous Sequencing Library Preparation of DNA and RNA Using Hydrogel Matrices.

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the standard protocols for generating genomic or transcriptomic libraries are incompatible and researchers must choose whether to sequence DNA or RNA for a particular sample. Gel-seq solves this problem by enabling researchers to simultaneously prepare libraries for both DNA and RNA starting with 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries. We designed Gel-seq so that it could be easily implemented by other researchers; many genetics labs already have the necessary equipment to reproduce the Gel-seq device fabrication. Our protocol employs commonly-used kits for both whole-transcript amplification (WTA) and library preparation, which are also likely to be familiar to researchers already versed in generating genomic and transcriptomic libraries. Our approach allows researchers to bring to bear the power of both DNA and RNA sequencing on a single sample without splitting and with negligible added cost.