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Detection and mutation surveillance of SARS-CoV-2 are crucial for combating the COVID-19 pandemic. Here we describe a lab-based method for multiplex isothermal amplification-based sequencing and real-time analysis of multiple viral genomes. It can simultaneously detect SARS-CoV-2, influenza A, human adenovirus, and human coronavirus and monitor mutations for up to 96 samples in real time. The method proved to be rapid and sensitive (limit of detection: 29 viral RNA copies/μL of extracted nucleic acid) in detecting SARS-CoV-2 in clinical samples. We expect it to offer a promising solution for rapid field-deployable detection and mutational surveillance of pandemic viruses.

The bioluminescent assay in real time (BART) is a molecular diagnostic tool for the detection of nucleic acid amplification by recording light output. The key component for BART is a thermostable luciferase derived from the firefly Photinus pyralis. Coupling BART detection with the isothermal amplification method loop-mediated isothermal amplification (LAMP) enables rapid molecular diagnostic results using simple equipment. LAMP-BART provides quantitative results from a closed tube and is appropriate to microliter standard tests and nanoliter microfluidic assays. In this chapter, we introduce a protocol to amplify and detect genetic markers using LAMP with BART. Furthermore, we provide advice to optimize LAMP assays for high sensitivity and specificity and to eliminate the incidence of "false positive" results which can occur from the components of the assay. The optimization of genetically modified (GM) maize by targeting the nopaline synthase terminator (NOSt) and 35S promoter (35Sp) sequences is described.

The ability to produce single-stranded DNA on a preparative scale from low amounts of starting templates is necessary for most research involving deoxyribozymes, but is particularly important for performing in vitro selections. While the production of single-stranded RNA is straightforward by means of in vitro transcription, the enzymatic production of single-stranded DNA (ssDNA) on a preparative scale is often difficult. Nevertheless, several methods for the production of ssDNA have been published over the years. Here, we present two PCR methods that we find to be particularly effective, fast, and affordable, which we have adapted for our own needs.

Loop-mediated isothermal amplification (LAMP) is a sensitive method that can rapidly amplify a specific nucleic acid target with high specificity. The LAMP reaction process has no denaturation step, instead DNA amplification occurs by strand displacement activity of the Bacillus stearothermophilus (Bst) DNA polymerase under isothermal conditions. It utilizes three sets of forward and reverse oligonucleotide primers specific to six distinct sequences on the target gene. These primers are used to generate amplification products that contain single-stranded loops, thereby allowing primers to bind to these sequences without the need for repeated cycles of thermal denaturation. For diagnosis of pathogens with RNA genome, LAMP has been merged with reverse transcription (RT) step to create RT-LAMP. To further reduce the cost of diagnosis and increase the throughput, immunocapture (IC) step was added to develop IC-RT-LAMP assay. Hence, this chapter focuses on utilizing IC-RT-LAMP assay to specifically identify severe strain of a plant virus from field samples.

A wide range of nucleic acid-based detection methods is available for viroid diagnosis. In this chapter, we describe a novel reverse transcription loop-mediated isothermal amplification (RT-LAMP). The technique involves the use of nucleic acids, plant sap, or even traces of plant tissue. Adding a set of four to six primers, Bst DNA polymerase, AMV, or another thermostable reverse transcriptase, allows the fast detection of RNA targets like viroids after a short (20-40 min) incubation at 63 °C either in an open or close-tube system.

One of the grand challenges for field-deployable NATs is related to the front end of the assays – nucleic acid extraction from raw samples. The ideal nucleic acid sample preparation should be simple, scalable, and easy-to-operate. In this chapter, we present a lab-on-a-disc NAT device for sample-to-answer malaria diagnosis. The parasite DNA sample preparation and subsequent real-time LAMP detection were seamlessly integrated on a disposable single microfluidic compact disc, driven by energy-efficient, non-centrifuge based magnetic field interactions. Each disc contains four parallel testing units, which could be configured either as four identical tests or as four species-specific tests. When configured as species-specific tests, it could identify two of the most life-threatening malaria species (P. falciparum and P. vivax). The reagent disc with a 4-plex analyzer (discussed in Chapter 1) is capable of processing four samples simultaneously with 40 min turnaround time. It achieves a detection limit of ~0.5 parasites/μl for whole blood, sufficient for detecting asymptomatic parasite carriers. The assay is performed with an automated device described in the companion chapter. The combination of sensitivity, specificity, cost, and scalable sample preparation suggests the real-time fluorescence LAMP device could be particularly useful for malaria screening in the field settings.

The point-of-care testing of DNA biomarkers requires compact biosensing systems and consumer electronic technologies provide fascinating opportunities. Their portability, mass-produced components, and high-performance readout capabilities are the main advantages for the development of novel bioanalytical methods.This chapter describes the detection of single nucleotide polymorphisms (SNP) through methods based on user-friendly optical devices (e.g., USB digital microscope, flatbed scanner, smartphone, and DVD drive). Loop mediated isothermal amplification (LAMP) enables the required discrimination of each specific variant prior to the optical reading. In the first method, products are directly hybridized to the allele-specific probes attached to plastic chips in an array format. The second method, allele-specific primers are used, enabling the direct end-point detection based a colorimetric dyer and a microfluidic chamber chip. In both approaches, devices are employed for chip scanning.A representative application to the genotyping of a clinically relevant SNP from human samples is provided, showing the excellent features achieved. Consumer electronic devices are able to register sensitive precise measurements in terms of signal-to-noise ratios, image resolution, and scan-to-scan reproducibility. The integrated DNA-based method lead a low detection limit (100 genomic DNA copies), reproducible (variation15%), high specificity (genotypes validated by reference method), and cheap assays (10 €/test). The underlying challenge is the reliable implementation into minimal-specialized clinical laboratories, incorporating additional advantages, such as user-friendly interface, low cost, and connectivity for telemedicine needs.

The FastPCR software is an integrated tool environment for PCR primer and probe design and for prediction of oligonucleotide properties. The software provides comprehensive tools for designing primers for most PCR and perspective applications, including standard, multiplex, long-distance, inverse, real-time with TaqMan probe, Xtreme Chain Reaction (XCR), group-specific, overlap extension PCR for multifragment assembling cloning, and isothermal amplification (Loop-mediated Isothermal Amplification). A program is available to design specific oligonucleotide sets for long sequence assembly by ligase chain reaction and to design multiplexed of overlapping and nonoverlapping DNA amplicons that tile across a region(s) of interest for targeted next-generation sequencing, competitive allele-specific PCR (KASP)-based genotyping assay for single-nucleotide polymorphisms and insertions and deletions at specific loci, among other features. The in silico PCR primer or probe search includes comprehensive analyses of individual primers and primer pairs. FastPCR includes various bioinformatics tools for analysis and searching of sequences, restriction I-II-III-type enzyme endonuclease analysis, and pattern searching. The program also supports the assembly of a set of contiguous sequences, consensus sequence generation, and sequence similarity and conservancy analysis. FastPCR performs efficient and complete detection of various repeat types with visual display. FastPCR allows for sequence file batch processing that is essential for automation. The software is available for download at https://primerdigital.com/fastpcr.html and online version at https://primerdigital.com/tools/pcr.html .

Several protocols for genomic amplification using reverse transcription followed by polymerase chain reaction (RT-PCR), important in the identification of the infecting serotype, have been used in the rapid diagnosis of Dengue Virus (DENV) infections. The qualitative protocol described by Lanciotti et al. (J Clin Microbiol 30: 545-551, 1992) suggested by WHO detects the four DENV serotypes simultaneously in one procedure "semi-nested," generating amplified products with specific sizes in base pairs for each serotype and it has been the most used in the past two decades. However, advances in molecular diagnosis have enabled the development of RT-PCR in real time (qRT-PCR) based on the use of dyes and probes (SYBR green and TaqMan), which is performed in a single step and is capable of providing quantitative data. In addition to quantification, the advantages of qRT-PCR over conventional RT-PCR include speed, greater sensitivity and specificity, and low rate of false positives. Several protocols for the diagnosis and/or quantification of DENV have already been described. Non-PCR-based methods such as reverse transcription loop-mediated isothermal amplification have shown high sensitivities and specificities. RT-PCR and qRT-PCR techniques can be performed using serum, plasma, infected cells, mosquitoes, fresh, and paraffin-embedded tissues. However, despite fast and accurate, they are limited to samples collected during the acute phase of infection (up to 7 days after the onset of symptoms) and require specialized equipment and trained staff.

Mycobacterium ulcerans is a slow-growing environmental bacterium that causes a severe skin disease known as Buruli ulcer (BU). Rapid detection of M. ulcerans in clinical specimens is essential for early diagnosis so that patients can be treated appropriately as soon as possible. This chapter describes suitable methods for the extraction of M. ulcerans DNA from the most common specimens submitted to the laboratory for confirmation of BU: swabs, fresh tissue biopsies, and fixed tissue sections. The resulting DNA extracts may be used for downstream procedures including standard gel-based PCR and real-time PCR assays. Protocols for direct detection of M. ulcerans DNA by real-time PCR are described in Chapter 8 .

The Ralstonia solanacearum species complex (RSSC) is composed of several Ralstonia species and strains that are little related and show varied host range and distinct geographic distributions. The RSSC causes wilt disease, and can thus have severe economic consequences for many important crops and ornamental plants. One such is potato (Solanum tuberosum), where infection causes brown rot of the tubers. It is important that symptomatic tubers and plants can be rapidly and easily tested, as exclusion of infected material is a cornerstone of management of bacterial diseases. A suitable method is loop-mediated isothermal amplification, a rapid, DNA-based method that can be used for specific detection of plant pathogens in infected materials. The combination of this loop-mediated isothermal amplification assay for the RSSC with a simple sample preparation method is fit for purpose for identification of this devastating disease in symptomatic tubers and plants. This methodology is rapid and cost efficient, and can be carried out outside of conventional laboratory facilities.

DNA methylation can regulate gene expression by modulating chromatin accessibility and transcription factor binding on promoter and enhancer regions. Whole-genome bisulfite sequencing (WGBS) represents the most informative and comprehensive analysis to profile the DNA methylation status of all the cytosines at single-base resolution. However, most of the available protocols recommend an amount of input DNA (50 ng-5μg) that makes the WGBS unsuitable for limited samples and cell populations. In this chapter, we provide complete protocol to perform WGBS libraries from very low-input DNA. This protocol is recommended for the analysis of the whole-genome DNA methylation pattern in rare cell populations, like a defined stem cell population isolated from animal models or human samples.

Active transposable elements (TEs) generate insertion polymorphisms that can be detected through genome resequencing strategies. However, these techniques may have limitations for organisms with large genomes or for somatic insertions. Here, we present a method that takes advantage of the extrachromosomal circular DNA (eccDNA) forms of actively transposing TEs in order to detect and characterize active TEs in any plant or animal tissue. Mobilome-seq consists in selectively amplifying and sequencing eccDNAs. It relies on linear digestion of genomic DNA followed by rolling circle amplification of circular DNA. Both active DNA transposons and retrotransposons can be identified using this technique.

ChIP-seq is a powerful technique that allows the detection of chromatin localization for proteins and epigenetic modifications. However, conventional ChIP-seq usually requires millions of cells. This becomes a daunting task for applications in which only limited experimental materials are available. For example, during mammalian embryo development, the epigenomes undergo drastic reprogramming which endows a fertilized egg with the potential to develop into the whole body. Low-input ChIP-seq methods would be instrumental to help decipher molecular mechanisms underlying such epigenetic reprogramming. Here we describe an optimized ChIP-seq method-STAR (Small-scale TELP-Assisted Rapid) ChIP-seq-that allows the detection of histone modifications using only a few hundred cells. This method is proven to be robust in epigenomic profiling in both embryos and cultured cells.

Developed by 3M Company, 3M ™ Molecular Detection Assays-3M MDS-enable detection of Salmonella from advanced isothermal DNA amplification and bioluminescence detection technology. It can be used for a wide variety of products, including poultry, eggs, pet foods, and environmental samples, and results are obtained within about 24 h. In this chapter, all steps of the 3M MDS™ method for detection of Salmonella are described and detailed.

Reverse transcription (RT) based quantitative PCR (qPCR) for quantifying microRNAs (miRNAs) in in the circulation presents specific challenges. Here, we describe an optimized research protocol to assess serum sample quality and quantify levels of a panel of four test miRNAs (miR-371a-3p, miR-372-3p, miR-373-3p, and miR-367-3p) that enables highly sensitive and specific malignant germ cell tumor (GCT) diagnosis and monitoring. This protocol utilizes a multiplex RT step using Taqman miRNA stem-loop primers. A multiplexed preamplification stage is then employed to increase the sensitivity of the final quantification step, which is performed using standard singleplex Taqman qPCR methodology.

Recent advances of image-based in situ mRNA quantification methods allow to visualize where in a tissue section a set of genes is expressed. It enables to map large numbers of genes in parallel and by capturing cellular boundaries allows to assign genes to cells. Here, we present a high-throughput, multi-targeted gene expression profiling technique called in situ sequencing that is capable of localizing hundreds of genes simultaneously and supports cell type classifications that follow transcriptome-based taxonomy. In situ sequencing is a targeted, amplified, and barcoded approach using padlock probes (PLPs) and rolling circle amplification (RCA). The current protocol relies on mRNA fixation, mRNA reverse transcription, residual mRNA degradation, and PLP hybridization. PLPs are amplified by RCA and labeled with fluorophore-conjugated probes, allowing their detection under conventional fluorescence microscopes.

Reverse Transcription-Loop-mediated Isothermal Amplification (RT-LAMP) allows amplification and detection of RNA or DNA rapidly and relatively inexpensively. Here we describe how RT-LAMP can be utilized to detect Zika virus in human urine or serum samples or Aedes mosquito samples. This can be completed in under 30 min and without first isolating the RNA from the sample.

Molecular detection of Zika virus (ZIKV) is a key element of outbreak management. Multiple PCR and isothermal ZIKV assays targeting different ZIKV sequences have been published. In this study, we compared a qRT-PCR, 2 RT-LAMP assays (based on different primer design approaches), and an RT-RPA for the detection of African and Asian/American lineages of ZIKV isolates from human, mosquito, and monkey. Results showed that RT-LAMP detected 100% of samples with a time threshold (Tt) of 18.01 ± 11.71 min while qRT-PCR detected 88.88% of samples with a Tt of 58.30 ± 16.58 min and RT-RPA 50% of samples with a Tt of 3.70 ± 0.44 min.

Development of novel point of care diagnostic methods in order to help in implementing disease control program and identifying the causative agent of an outbreak is crucial. Classical diagnostic techniques, e.g., real-time polymerase chain reaction (PCR), rely on the presence of the nucleic acid sequence of the target in GenBank. In the case of an emerging new strain of a known or novel pathogen, false-negative results will be recorded by PCR. On the other hand, next-generation sequencing technologies allow rapid whole genome sequencing without previous knowledge of the target. One of these methods is the Oxford Nanopore sequencing technique, which utilizes a portable device named MinION and has a short run time. In this protocol, we describe the development of a novel nanopore sequencing protocol by combining random isothermal amplification technology and nanopore sequencing. The established protocol is rapid (7 h) and sensitive as less than 4% of the sequenced RNA belonged to the target virus, Zika. Interestingly, we have established an offline BLAST search for the data analysis that facilitates the use of the whole protocol at remote settings without the need of an Internet connection.

Two one-step real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for the detection of Zika virus (ZIKV) were developed, based on two different primer design approaches: (1) open source, based on a combination of sequence diversity clustering (phylogeny and principal component analysis) and LAVA algorithm, using 45 whole genome ZIKV sequences retrieved from the National Center for Biotechnology Information (NCBI) database; (2) standard software for LAMP primer design (Primer Explorer V4), using 59 sequences of the ZIKV 3' UTR. The assays were firstly evaluated with External Quality Assessment panels from INSTAND e.V. (Germany) and EVD-LabNet (The Netherlands) including 4 and 12 unknown samples, respectively, and secondly, with 9 human, mosquito, and monkey ZIKV isolates from Africa (Senegal, Ivory Coast, and Uganda) and America (Brazil). The limit of detection as determined by probit analysis was 181 molecules for both RT-LAMP assays, and 100% reproducibility in the assays was obtained for 10

Spatial genome organization in the cell nucleus plays a crucial role in the control of genome functions. Our knowledge about spatial genome organization is relying on the advances in gene imaging technologies and the biochemical approaches based on the spatial dependent ligation of the genomic regions. Fluorescent in situ hybridization using specific fluorescent DNA and RNA probes in cells and tissues with the spatially preserved nuclear and genome architecture (3D-FISH) provides a powerful tool for the further advancement of our knowledge about genome structure and functions. Here we describe the 3D-FISH protocols allowing for such an analysis in mammalian tissue in situ including in the skin. These protocols include DNA probe amplification and labeling; tissue fixation; preservation and preparation for hybridization; hybridization of the DNA probes with genomic DNA in the tissue; and post-hybridization tissue sample processing.

This chapter reports a library preparation protocol for efficient high-throughput sequencing of double-stranded RNA viruses. The protocol consists of four main steps, viz., enzyme treatment, precipitation using lithium chloride, full-length amplification of cDNAs, and tailing adapters for high-throughput sequencing. This protocol will be useful for all double-stranded RNA viruses and for all of the high-throughput sequencing platforms.

Rolling circle amplification (RCA) of a synthetic nucleic acid target is detected using magnetic nanoparticles (MNPs) combined with an optomagnetic (OM) readout. Two RCA assays are developed with on-chip detection of rolling circle products (RCPs) either at end-point where MNPs are mixed with the sample after completion of RCA or in real time where MNPs are mixed with the sample during RCA. The plastic chip acts as a cuvette, which is positioned in a setup integrated with temperature control and simultaneous detection of four parallel DNA hybridization reactions between functionalized MNPs and products of DNA amplification. The OM technique probes the small-angle rotation of MNPs bearing oligonucleotide probes complementary to the repeated nucleotide sequence of the RCPs. This rotation is restricted when MNPs bind to RCPs, which can be observed as a turn-off of the signal from MNPs that are free to rotate. The amount of MNPs bound to RCPs is found to increase in response to the amplification time as well as in response to the synthetic DNA target concentration (2-40 pM dynamic range). We report OM real-time results obtained with MNPs present during RCA and compare to relevant end-point OM results for RCPs generated for different RCA times. The real-time approach avoids opening of tubes post-RCA and thus reduces risk of lab contamination with amplification products without compromising the sensitivity and dynamic range of the assay.

Next generation sequencing (NGS) and assembly are key pieces in the success of cereal crops genomes sequencing. One interesting strategy for reducing the complexity in large genomes, the case of several cereal crops, is the sequencing of individual chromosomes. This has been done with success by flow cytometric chromosome sorting followed by sequencing using available next generation (high throughput) sequencing platforms. In this chapter, methodologies for sequencing and assembly of flow sorted chromosomes and whole genomes in cereal crops, with special emphasis on the case of the International Wheat Genome Sequencing Consortium (IWGSC), are reviewed.

High-throughput sequencing of the products of 5' RNA ligase-mediated rapid amplification of cDNA ends (5' RLM-RACE) reactions (RACE-SEQ) enables the mapping and digital enumeration of expected and novel 5' ends in RNA molecules. The resulting data are essential in documenting the mechanism of action and precision of endonucleolytically active, RNA-targeting drugs such as RNase H-active antisense or small interfering RNA. When applied to error-prone replication systems such as RNA viruses or in vitro RNA replicon systems, the method can additionally report the relative susceptibility of known and unknown polymorphisms to a prospective sequence-specific drug, making it a powerful tool in patient selection and stratification as well as resistance prediction.We describe the preparation of sequencing libraries for ultra-high depth 5' RLM-RACE analysis on two popular second-generation high-throughput sequencing platforms (Illumina, Ion Torrent) and supply a detailed bioinformatics analysis pipeline for target site activity definition and enumeration. We further illustrate how the pipeline can be simply modified to generate polymorphism-specific drug susceptibility data from in vitro replicon experiments (RACE-SEQ-MM), in a patient-free manner, to cover both known and unknown target site variants in the population.

Loop-mediated isothermal amplification (LAMP) is a new nucleic acid amplification technique that provides high sensitivity and specificity compatible to conventional polymerase chain reaction (PCR) and simplicity, without the requirement of an expensive thermal cycler. Additionally, LAMP coupled hydroxynaphthol blue (HNB) allowed for visual color detection by the naked eye. Here we describe the methodology of Chlamydia trachomatis detection of vaginal swab from crude DNA lysis to LAMP-HNB reaction. The result can be read by the naked eye through color change from violet (negative) to sky blue (positive). The result can be confirmed by UV spectra and agarose gel electrophoresis. This assay uses all nonhazardous chemical reagents and is hence safe to the users, and requires little specialist training or knowledge.

The loop-mediated isothermal amplification (LAMP) is one of the most widely used isothermal nucleic acid amplification techniques due to it its simplicity and adaptability within limited resource or point-of-care settings. Here, LAMP was utilized for the rapid amplification and detection of Chlamydia trachomatis and Neisseria gonorrhoeae.

Detection of any decrease in telomerase activity in cancer cells and tumor tissues is an important part in assessing overall therapeutic outcomes of a treatment agent in the laboratory and clinical settings. Almost 85% of cancers have activation of telomerase activity that promotes cell proliferation and discourages differentiation to sustain growth of the cancers. Retinoids are highly regarded as the anti-proliferation and pro-differentiation agents that cause down regulation of telomerase activity in the cancer cells. Two (nonradioactive and radioactive) telomeric repeat amplification protocol (TRAP) assays are optimized and fully described for detection of the diminished or abolished telomerase activity in a very low amount of protein extracts from cancer cells after treatment with a natural retinoid or a synthetic retinoid. These highly optimized and improved nonradioactive and radioactive TRAP assays can also be used for determining the presence or absence of telomerase activity in a small amount of any tumor tissue. The results from these TRAP assays can also help decide appropriate therapeutic options for the cancers with or without telomerase activity.

Loop-mediated isothermal amplification (LAMP) is one recently developed gene amplification technique that emerges as a simple and quick diagnostic tool for early detection of nucleic acid targets. The LAMP technique works on the principle of strand displacement activity of Bst polymerase. It contains a set of four specially designed primers, which recognizes six different regions on the target nucleotide sequence. In the LAMP reaction, amplification is carried out in an isothermal conditions (60-65°C) using simple and inexpensive device like water bath or dry bath. Additional benefits of LAMP technique are that final results can be seen directly with naked eyes by adding intercalating dye SYBR Green I in the reaction tube. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is one of the novel techniques used for detection of RNA targets. The technology has been successfully applied for rapid and sensitive detection of Citrus tristeza virus (CTV) by using four oligo-primers, targeting a conserved coat protein gene (CPG) of an Indian CTV isolate. The result of assay is visible in naked eyes easily in the presence of SYBR Green I (100×) or on 1.5% agarose gel electrophoresis. CTV-RT-LAMP could be used away from plant pathology laboratories even in remote location.

Treatment trials of antibiotics for Neisseria gonorrhoeae infections frequently enroll primarily men with urethritis, as the diagnosis of acute gonococcal infection in men with urethritis is easily made by Gram stain of the urethral exudate, followed by confirmatory culture or nucleic acid amplification tests (NAATs). Enrolling women in treatment trials is of great importance, but N. gonorrhoeae cervical infections cause nonspecific symptoms. This makes it difficult to conduct interventional trials, as large numbers of women with nonspecific symptoms need to be screened for infection. Gram stain of cervical secretions has a strikingly low sensitivity, and culture and/or NAAT results are not available at the time of screening. This necessitates recall and delayed treatment of infected women who may not return and who may spread the infection during the interval. In this chapter we present an algorithm, derived from a comparison of women who did, or did not, become infected during exposure, which identifies those women who are highly likely to be infected before culture and/or NAAT results are available. The algorithm provides an efficient way to conduct interventional trials in women without the problem of recall and delayed treatment.

Single-cell RNA sequencing has revolutionized the way we look at cell populations. Of the methods available, CEL-Seq was the first to use linear RNA amplification. With early barcoding and 3' sequencing, it is sensitive, cost-effective and easy to perform. Here we describe a protocol for performing CEL-Seq2 on sorted cells, which can be performed without any special equipment.

Single-cell genome sequencing can detect low-frequency genetic alterations present in complex tissues. However, the experimental procedures are technically challenging. This includes dissociation of the tissue, isolation of single cells, whole-genome amplification, sequencing library preparation, and an optional target enrichment. Here we describe how to perform each of these processes to obtain high-quality single-cell genome sequencing data.

All organisms release their DNA into the environment through processes such as excretion and the senescence of tissues and limbs. This DNA, often referred to as environmental DNA (eDNA) or sedimentary ancient DNA (sedaDNA), can be recovered from both present-day and ancient soils, fecal samples, bodies of water and lake cores, and even air. While eDNA is a potentially useful record of past and present biodiversity, several challenges complicate data generation and interpretation of results. Most importantly, eDNA samples tend to be highly taxonomically mixed, and the target organism or group of organisms may be present at very low abundance within this mixture. To overcome this challenge, enrichment approaches are often used to target specific taxa of interest. Here, we describe a protocol to amplify metabarcodes or short, variable loci that identify lineages within broad taxonomic groups (e.g., plants, mammals), using the polymerase chain reaction (PCR) with established generic "barcode" primers. We also provide a catalog of animal and plant barcode primers that, because they target relatively short fragments of DNA, are potentially suitable for use with degraded DNA.

The use of direct nucleic acid amplification of pathogens from food matrices has the potential to reduce time to results over DNA extraction-based approaches as well as traditional culture-based approaches. Here we describe protocols for assay design and experiments for direct amplification of foodborne pathogens in food sample matrices using loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR). The examples provided include the detection Escherichia coli in milk samples and Salmonella in pork meat samples. This protocol includes relevant reagents and methods including obtaining target sequences, assay design, sample processing, and amplification. These methods, though used for specific example matrices, could be applied to many other foodborne pathogens and sample types.

Genome diversity comprises single nucleotide polymorphisms, deletions, insertions, and duplications. These gains and losses of DNA segments leading to rearrangements of sequences are termed copy number variations (CNVs). CNVs may disrupt genes and/or alter gene dosage and, thereby, have an impact on both protein-coding and noncoding genes. Accordingly, they affect the activity of various signaling pathways and influence the cell phenotype. They are associated with risks for several severe diseases, in particular cancer. In the current chapter, I introduce a rapid profiling method to identify CNVs in circulating, cell-free DNA by multiplex ligation-dependent probe amplification (MLPA). MLPA represents an efficient method for the detection of CNVs among numerous genes on various chromosomal regions in serum.

Whole-exome sequencing (WES) has been used as a standard of care for postnatal diagnosis in the clinical setting in the past few years for children and adults with undiagnosed disease. Many rare disorders have been diagnosed through WES, which is less expensive than the traditional serial genetic testing where patients had previously spent years on an uninformative diagnostic odyssey. Seeking a diagnosis often entails enduring time consuming, and sometimes invasive procedures which may be associated with medical risks that are stressful for families and impose a heavy burden on the health-care system. However, the use of WES is considered impractical in the prenatal and neonatal testing period because of the technical and computational challenges of performing genomic sequencing from small amounts of genetic material, and the need for faster turnaround time (TAT) than the current 6-8 weeks TAT provided by most clinical labs offering postnatal testing. With the rapidly evolving methods of sequence analysis, there are clinical challenges such as the constantly increasing number of genes being identified which are not yet fully phenotypically characterized, especially when ascertained prenatally or neonatally before all the clinical features may be evident. Despite these challenges, there are many clinical benefits to acquiring genomic information in the prenatal and neonatal period. These include superior prognostic information which allows for prenatal planning of mode of delivery and hospital for delivery and optimized neonatal management. We have developed a clinical WES assay using small amounts of DNA with a TAT of 10 days for use in the prenatal or neonatal setting. This test is used to detect small nucleotide variants and indels in fetuses and neonates with structural abnormalities.

Multiplex Ligation-dependent Probe Amplification (MLPA) is a method to determine the copy number of up to 60 genomic DNA sequences in a single multiplex PCR based reaction.MLPA probes consist of two oligonucleotides that can hybridize next to each other on a certain DNA sequence of interest, where they are ligated. All ligated probes are subsequently amplified by PCR using a single set of primers. Each amplified MLPA probe has a unique length and can be visualized and quantified by capillary electrophoresis. As the primers are almost 100% consumed in the PCR reaction, the quantity of each PCR amplicon is proportional to the number of copies of each probe target sequence in the DNA sample. A trisomy 21 can therefore be detected by an approximately 50% increased signal of each chromosome 21 specific probe relative to reference samples.MLPA with the P095 Aneuploidy probemix for chromosomes 13, 18, 21, X and Y has been used as a rapid detection method on large numbers of samples from uncultured amniotic fluid or from chorionic villi. As compared to FISH and karyotyping, MLPA is more rapid, has a higher throughput, and is less expensive. MLPA however cannot detect low grade mosaicism, female triploidies, and copy number neutral chromosome abnormalities such as inversions and translocations.

The human urinary virome is comprised by diverse communities of both eukaryotic viruses and bacteriophages. Identification and quantification of human urinary viruses was limited to PCR-based and culture methods. Detection of viruses in human urine has long been associated with disease, but advances in next-generation sequencing have shown that it harbors robust viral communities. Human urinary viral communities are mainly comprised by bacteriophages and Human Papillomaviruses (HPVs) that remain to be characterized in association with health and diverse urinary disease states. In the present chapter, the methods to identify and quantify DNA viral populations in human urine using next-generation sequencing are described.

The protocol presented here allows the isolation, purification, nucleic acid extraction, and amplification of DNA/RNA from viruses present in human sera samples. The method allows the random amplification of the viral genomes present by using a Sequence-Independent, Single-Primer Amplification (SISPA) approach enabling the study of both DNA/RNA viruses. An amplification step is needed, as the concentration of viral DNA/RNA in serum samples is low for direct library preparation. The application of the described protocol guarantees enough randomly amplified double-strand DNA for further library preparation using Nextera XT kit from Illumina.

Virome studies are of special interest nowadays. Understanding viral communities in different body compartments will help guide future personalized treatments and to discern between homeostasis and disease. High-throughput sequencing technologies allow us to detect all the nucleic acids present in a sample, including viral ones, by random sequencing. One of the major challenges in virome studies is the correct isolation of the viral nucleic acids from a specific sample. This can be done during the extraction steps (e.g., enrichment of viral capsids), or during the bioinformatic analysis (e.g., removing all human and bacterial sequences). Furthermore, it is an important remark that the treatment of the sample will strongly influence the results. Samples will be treated differently if the ultimate goal is the study of all replicating and encapsidated viruses, including both RNA and DNA ones, if we are only focused on DNA ones, or if we want to analyze all the possible viral nucleic acids in the specific sample, even if the genome is degraded. Here, we present a technique that allows for isolation of viral nucleic acids from plasma samples.

In order to describe a novel uncultured viral species, it is essential to obtain the DNA sequence of their full genomes. The ability to distinguish the viral genome from the genome of its bacterial host is the major challenge of the modern viromics. The major obstacles for mining of viral genomes in metagenomic assemblies is bacterial contamination in viromes and low DNA input for sequencing.These obstacles can be overcome by flow cytometry that allows collecting free viral particles from environmental samples. In addition, fluorescence activated cell sorting reduces the bacterial contamination. By using optimized sequencing protocols, the ultra-low input DNA samples can be sequenced directly, without the need for whole genome amplification. This chapter provides details for staining of environmental viruses, flow cytometry, and direct sequencing of ultra-low input DNA samples on Illumina platform.

TILLING is a reverse genetics strategy that combines the high density of point mutations provided by traditional chemical mutagenesis with rapid screening of DNA pools from a mutagenized population for induced mutations (McCallum et al., Nat Biotechnol 18:455-457, 2000). This high-throughput technique allows the identification of point mutations in any gene of interest.

RNA sequencing is a recently developed approach for transcriptome profiling with several advantages over gene expression profiling using DNA microarrays. Here we describe a RNA-sequencing protocol optimized for low-input analysis of total RNA from CD138

Central nervous system (CNS) disease caused by Mycobacterium tuberculosis (MTB) is highly devastating. Tuberculous meningitis (TBM) is the most common form of CNS tuberculosis (TB). Rapid, sensitive, and affordable diagnostic tests are not available. Ziehl-Neelsen (ZN) stain has a very low sensitivity in cases of TBM, the sensitivity rates is of about 10-20%.The detection rate can be improved by taking large volume CSF samples (6 ml) and prolonged slide examination (30 min). Culture of MTB from the CSF is slow and insufficiently sensitive. The sensitivity is different, which varies from 36% to 81.8%. The microscopic observation drug susceptibility (MODS) assay was recommended by the World Health Organization in 2011. The sensitivity is 65%, which is more sensitive and faster than CSF smear. Commercial PCR assays were found to be insensitive at detecting MTB in CSF samples. Many research provided the value of ADA on the TBM diagnosis. Interferon-gamma release assays (IGRAs) are not recommended for diagnosis of active TB disease. Imaging is essential in diagnosis and showing complications of CNS TB. Thwaites criteria and the Lancet consensus scoring system (LCSS) were developed to improve the diagnosis of TBM. Clinicians will continue to make judgment based on clinical examination, inflammatory CSF examinations, imaging studies, and scoring systems.

High-throughput sequencing (or next-generation sequencing-NGS) is an emerging technology that allows the detection of plant viruses without any prior knowledge. Various sequencing techniques and various templates can be used as substrate for NGS. This chapter describes an optimized protocol for the extraction of double-stranded RNAs (dsRNAs) from a wide range of plants and for their random amplification prior to NGS sequencing.

In this chapter, we describe the use of Illumina

Single-cell RNA sequencing (sc-RNASeq) is a recently developed technique used to evaluate the transcriptome of individual cells. As opposed to conventional RNASeq in which entire populations are sequenced in bulk, sc-RNASeq can be beneficial when trying to better understand gene expression patterns in markedly heterogeneous populations of cells or when trying to identify transcriptional signatures of rare cells that may be underrepresented when using conventional bulk RNASeq. In this method, we describe the generation and analysis of cDNA libraries from single patient-derived glioblastoma cells using the C1 Fluidigm system. The protocol details the use of the C1 integrated fluidics circuit (IFC) for capturing, imaging and lysing cells; performing reverse transcription; and generating cDNA libraries that are ready for sequencing and analysis.

Next-generation small RNA sequencing is a valuable tool which is increasing our knowledge regarding small noncoding RNAs and their function in regulating genetic information. Library preparation protocols for small RNA have thus far been restricted due to higher RNA input requirements (10 ng), long workflows, and tedious manual gel purifications. Small RNA library preparation methods focus largely on the prevention or depletion of a side product known as adapter dimer that tends to dominate the reaction. Adapter dimer is the ligation of two adapters to one another without an intervening library RNA insert or any useful sequencing information. The amplification of this side reaction is favored over the amplification of tagged library since it is shorter. The small size discrepancy between these two species makes separation and purification of the tagged library very difficult. Adapter dimer hinders the use of low input samples and the ability to automate the workflow so we introduce an improved library preparation protocol which uses chemically modified adapters (CleanTag) to significantly reduce the adapter dimer. CleanTag small RNA library preparation workflow decreases adapter dimer to allow for ultra-low input samples (down to approx. 10 pg total RNA), elimination of the gel purification step, and automation. We demonstrate how to carry out this streamlined protocol to improve NGS data quality and allow for the use of sample types with limited RNA material.