DeLTa-Seq: direct-lysate targeted RNA-Seq from crude tissue lysate

AbstractUsing current mRNA quantification methods such as RT-qPCR and RNA-Seq, it is very difficult to examine thousands of tissue samples due to cost and labor of RNA extraction and quantification steps. Here, we developed Direct-RT buffer in which homogenization of tissue samples and direct-lysate reverse transcription can be conducted without RNA purification. We showed that appreciate concentration of DTT prevented RNA degradation but not RT in the lysates of several plants’ tissues, yeast, and zebrafish larvae. Using the buffer, direct reverse transcription on the lysates could produce comparable amount of cDNA with that synthesized from purified RNA. Furthermore, we established DeLTa-Seq (Direct-Lysate reverse transcription and Targeted RNA-Seq) method. DeLTa-Seq is a cost-effective, high-throughput and highly-precise quantification method for the expressions of hundreds of genes. It enables us to conduct large-scale studies using thousands of samples such as chemical screening, field experiments and studies focusing on individual variability.

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SARS-CoV-2 RNA Extraction Using Magnetic Beads for Rapid Large-Scale Testing by RT-qPCR and RT-LAMP

Viruses ◽

10.3390/v12080863 ◽

2020 ◽

Vol 12 (8) ◽

pp. 863 ◽

Cited By ~ 2

Author(s):

Steffen Klein ◽

Thorsten G. Müller ◽

Dina Khalid ◽

Vera Sonntag-Buck ◽

Anke-Mareil Heuser ◽

...

Keyword(s):

Reverse Transcription ◽

Large Scale ◽

Rna Extraction ◽

Magnetic Bead ◽

Viral Rna ◽

Detection Sensitivity ◽

Detection Methods ◽

N Gene ◽

Extraction Protocol ◽

Large Scale Testing

Rapid large-scale testing is essential for controlling the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The standard diagnostic pipeline for testing SARS-CoV-2 presence in patients with an ongoing infection is predominantly based on pharyngeal swabs, from which the viral RNA is extracted using commercial kits, followed by reverse transcription and quantitative PCR detection. As a result of the large demand for testing, commercial RNA extraction kits may be limited and, alternatively, non-commercial protocols are needed. Here, we provide a magnetic bead RNA extraction protocol that is predominantly based on in-house made reagents and is performed in 96-well plates supporting large-scale testing. Magnetic bead RNA extraction was benchmarked against the commercial QIAcube extraction platform. Comparable viral RNA detection sensitivity and specificity were obtained by fluorescent and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) using a primer set targeting the N gene, as well as RT-qPCR using a primer set targeting the E gene, showing that the RNA extraction protocol presented here can be combined with a variety of detection methods at high throughput. Importantly, the presented diagnostic workflow can be quickly set up in a laboratory without access to an automated pipetting robot.

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A Robust Metatranscriptomic Technology for Population-Scale Studies of Diet, Gut Microbiome, and Human Health

International Journal of Genomics ◽

10.1155/2019/1718741 ◽

2019 ◽

Vol 2019 ◽

pp. 1-9 ◽

Cited By ~ 1

Author(s):

Andrew Hatch ◽

James Horne ◽

Ryan Toma ◽

Brittany L. Twibell ◽

Kalie M. Somerville ◽

...

Keyword(s):

Human Health ◽

Gut Microbiome ◽

Large Scale ◽

Rna Extraction ◽

Rna Degradation ◽

Small Scale ◽

Sample Collection ◽

Precise Control ◽

Wide Range ◽

Population Scale

A functional readout of the gut microbiome is necessary to enable precise control of the gut microbiome’s functions, which support human health and prevent or minimize a wide range of chronic diseases. Stool metatranscriptomic analysis offers a comprehensive functional view of the gut microbiome, but despite its usefulness, it has rarely been used in clinical studies due to its complexity, cost, and bioinformatic challenges. This method has also received criticism due to potential intrasample variability, rapid changes, and RNA degradation. Here, we describe a robust and automated stool metatranscriptomic method, called Viomega, which was specifically developed for population-scale studies. Viomega includes sample collection, ambient temperature sample preservation, total RNA extraction, physical removal of ribosomal RNAs (rRNAs), preparation of directional Illumina libraries, Illumina sequencing, taxonomic classification based on a database of >110,000 microbial genomes, and quantitative microbial gene expression analysis using a database of ~100 million microbial genes. We applied this method to 10,000 human stool samples and performed several small-scale studies to demonstrate sample stability and consistency. In summary, Viomega is an inexpensive, high-throughput, automated, and accurate sample-to-result stool metatranscriptomic technology platform for large-scale studies and a wide range of applications.

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ApharSeq: An Extraction-free Early-Pooling Protocol for Massively Multiplexed SARS-CoV-2 Detection

10.1101/2020.08.08.20170746 ◽

2020 ◽

Author(s):

Alon Chappleboim ◽

Daphna Joseph-Strauss ◽

Ayelet Rahat ◽

Israa Sharkia ◽

Miriam Adam ◽

...

Keyword(s):

Reverse Transcription ◽

Limit Of Detection ◽

Massively Parallel Sequencing ◽

Rna Extraction ◽

Clinical Samples ◽

Rna Seq ◽

Reverse Transcription Pcr ◽

Sample Pooling ◽

Next Generation Sequencing Ngs ◽

Multiple Pathogens

The global SARS-CoV-2 pandemic created a dire need for viral detection tests worldwide. Most current tests for SARS-CoV-2 are based on RNA extraction followed by quantitative reverse-transcription PCR assays. While automation and improved logistics increased the capacity of these tests, they cannot exceed the lower bound dictated by one extraction and one RT-PCR reaction per sample. Multiplexed next generation sequencing (NGS) assays provide a dramatic increase in throughput, and hold the promise of richer information including viral strains, host immune response, and multiple pathogens. Here, we establish a significant improvement of existing RNA-seq detection protocols. Our workflow, ApharSeq, includes a fast and cheap RNA capture step, that is coupled to barcoding of individual samples, followed by sample-pooling prior to the reverse transcription, PCR and massively parallel sequencing. Thus, only one non-enzymatic step is performed before pooling hundreds of barcoded samples for subsequent steps and further analysis. We characterize the quantitative aspects of the assay by applying ApharSeq to more than 500 clinical samples in a robotic workflow. The assay results are linear, and the empirical limit of detection is found to be Ct 33 (roughly 1000 copies/ml). A single ApharSeq test currently costs under 1.2$, and we estimate costs can further go down 3-10 fold. Similarly, we estimate a labor reduction of 10-100 fold, automated liquid handling of 5-10 fold, and reagent requirement reduction of 20-1000 fold compared to existing testing methods.

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A robust metatranscriptomic technology for population-scale studies of diet, gut microbiome, and human health

10.1101/659615 ◽

2019 ◽

Author(s):

Andrew Hatch ◽

James Horne ◽

Ryan Toma ◽

Brittany L. Twibell ◽

Kalie M. Somerville ◽

...

Keyword(s):

Human Health ◽

Gut Microbiome ◽

Large Scale ◽

Rna Extraction ◽

Rna Degradation ◽

Small Scale ◽

Sample Collection ◽

Precise Control ◽

Wide Range ◽

Population Scale

ABSTRACTA functional readout of the gut microbiome is necessary to enable precise control of the gut microbiome’s functions, which support human health and prevent or minimize a wide range of chronic diseases. Stool metatranscriptomic analysis offers a comprehensive functional view of the gut microbiome, but despite its usefulness, it has rarely been used in clinical studies due to its complexity, cost, and bioinformatic challenges. This method has also received criticism due to potential intra-sample variability, rapid changes, and RNA degradation. Here, we describe a robust and automated stool metatranscriptomic method, called Viomega, which was specifically developed for population-scale studies. Viomega includes sample collection, ambient temperature sample preservation, total RNA extraction, physical removal of ribosomal RNAs (rRNAs), preparation of directional Illumina libraries, Illumina sequencing, taxonomic classification based on a database of >110,000 microbial genomes, and quantitative microbial gene expression analysis using a database of ~100 million microbial genes. We applied this method to 10,000 human stool samples, and performed several small-scale studies to demonstrate sample stability and consistency. In summary, Viomega is an inexpensive, high throughput, automated, and accurate sample-to-result stool metatranscriptomic technology platform for large-scale studies and a wide range of applications.

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Single cell high-throughput qRT-PCR protocol

10.21203/rs.3.pex-919/v1 ◽

2021 ◽

Author(s):

Sirisha Achanta ◽

Rajanikanth Vadigepalli

Keyword(s):

Single Cell ◽

High Throughput ◽

Reverse Transcription ◽

Single Cells ◽

Rna Extraction ◽

High Sensitivity ◽

Single Experiment ◽

Tissue Samples ◽

Qrt Pcr ◽

Cell Sample

Abstract Single cell high-throughput qRT-PCR protocol combines high sensitivity technique of single cell qPCR with high-throughput qPCR technology that can generate data from 96 samples and 96 genes in a single experiment. It can be adapted for various sample types- cell culture, tissue samples and extracted RNA (10 pg) and measured on traditional qPCR and high-throughput qPCR platforms. The workflow is comprised of four steps – cell lysis, reverse transcription, pre-amplification and qPCR. Key features of this protocol are; processing low input samples directly to reverse transcription without RNA extraction which minimizes sample loss, pre-amplification enables amplification of cDNA from single cells to detectable levels for qPCR and measuring up to 400 genes from a single cell sample/10 pg of RNA (starting material). Robust, reproducible and versatile this protocol can be adapted to several upstream and downstream techniques.

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Isolation of Nuclei in Tagged Cell Types (INTACT), RNA Extraction and Ribosomal RNA Degradation to Prepare Material for RNA-Seq

BIO-PROTOCOL ◽

10.21769/bioprotoc.2458 ◽

2018 ◽

Vol 8 (7) ◽

Cited By ~ 2

Author(s):

Mauricio Reynoso ◽

Germain Pauluzzi ◽

Sean Cabanlit ◽

Joel Velasco ◽

Jérémie Bazin ◽

...

Keyword(s):

Ribosomal Rna ◽

Rna Extraction ◽

Cell Types ◽

Rna Degradation ◽

Rna Seq

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Transcriptional and morphological profiling of parvalbumin interneuron subpopulations in the mouse hippocampus

Nature Communications ◽

10.1038/s41467-020-20328-4 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Lin Que ◽

David Lukacsovich ◽

Wenshu Luo ◽

Csaba Földy

Keyword(s):

Large Scale ◽

Cell Types ◽

Rna Seq ◽

Neuronal Identity ◽

Parvalbumin Interneurons ◽

Different Types ◽

Parvalbumin Interneuron ◽

Cam Profile ◽

Developmental Domains

AbstractThe diversity reflected by >100 different neural cell types fundamentally contributes to brain function and a central idea is that neuronal identity can be inferred from genetic information. Recent large-scale transcriptomic assays seem to confirm this hypothesis, but a lack of morphological information has limited the identification of several known cell types. In this study, we used single-cell RNA-seq in morphologically identified parvalbumin interneurons (PV-INs), and studied their transcriptomic states in the morphological, physiological, and developmental domains. Overall, we find high transcriptomic similarity among PV-INs, with few genes showing divergent expression between morphologically different types. Furthermore, PV-INs show a uniform synaptic cell adhesion molecule (CAM) profile, suggesting that CAM expression in mature PV cells does not reflect wiring specificity after development. Together, our results suggest that while PV-INs differ in anatomy and in vivo activity, their continuous transcriptomic and homogenous biophysical landscapes are not predictive of these distinct identities.

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Development and validation of a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of ZIKV in patient samples from Brazil

Scientific Reports ◽

10.1038/s41598-021-83371-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Severino Jefferson Ribeiro da Silva ◽

Keith Pardee ◽

Udeni B. R. Balasuriya ◽

Lindomar Pena

Keyword(s):

Reverse Transcription ◽

Rapid Detection ◽

Quantitative Polymerase Chain Reaction ◽

Rna Extraction ◽

Lamp Assay ◽

Isothermal Amplification ◽

Serum Samples ◽

Low Resource ◽

Loop Mediated Isothermal Amplification ◽

One Step

AbstractWe have previously developed and validated a one-step assay based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of the Zika virus (ZIKV) from mosquito samples. Patient diagnosis of ZIKV is currently carried out in centralized laboratories using the reverse transcription-quantitative polymerase chain reaction (RT-qPCR), which, while the gold standard molecular method, has several drawbacks for use in remote and low-resource settings, such as high cost and the need of specialized equipment. Point-of-care (POC) diagnostic platforms have the potential to overcome these limitations, especially in low-resource countries where ZIKV is endemic. With this in mind, here we optimized and validated our RT-LAMP assay for rapid detection of ZIKV from patient samples. We found that the assay detected ZIKV from diverse sample types (serum, urine, saliva, and semen) in as little as 20 min, without RNA extraction. The RT-LAMP assay was highly specific and up to 100 times more sensitive than RT-qPCR. We then validated the assay using 100 patient serum samples collected from suspected cases of arbovirus infection in the state of Pernambuco, which was at the epicenter of the last Zika epidemic. Analysis of the results, in comparison to RT-qPCR, found that the ZIKV RT-LAMP assay provided sensitivity of 100%, specificity of 93.75%, and an overall accuracy of 95.00%. Taken together, the RT-LAMP assay provides a straightforward and inexpensive alternative for the diagnosis of ZIKV from patients and has the potential to increase diagnostic capacity in ZIKV-affected areas, particularly in low and middle-income countries.

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Large-scale transcriptomics to dissect 2 years of the life of a fungal phytopathogen interacting with its host plant

BMC Biology ◽

10.1186/s12915-021-00989-3 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Elise J. Gay ◽

Jessica L. Soyer ◽

Nicolas Lapalu ◽

Juliette Linglin ◽

Isabelle Fudal ◽

...

Keyword(s):

Host Plant ◽

Large Scale ◽

Field Experiments ◽

Plant Residues ◽

Plant Infection ◽

Effector Genes ◽

Controlled Conditions ◽

Niche Adaptation ◽

Depth Analysis ◽

Fungal Genes

Abstract Background The fungus Leptosphaeria maculans has an exceptionally long and complex relationship with its host plant, Brassica napus, during which it switches between different lifestyles, including asymptomatic, biotrophic, necrotrophic, and saprotrophic stages. The fungus is also exemplary of “two-speed” genome organisms in the genome of which gene-rich and repeat-rich regions alternate. Except for a few stages of plant infection under controlled conditions, nothing is known about the genes mobilized by the fungus throughout its life cycle, which may last several years in the field. Results We performed RNA-seq on samples corresponding to all stages of the interaction of L. maculans with its host plant, either alive or dead (stem residues after harvest) in controlled conditions or in field experiments under natural inoculum pressure, over periods of time ranging from a few days to months or years. A total of 102 biological samples corresponding to 37 sets of conditions were analyzed. We show here that about 9% of the genes of this fungus are highly expressed during its interactions with its host plant. These genes are distributed into eight well-defined expression clusters, corresponding to specific infection lifestyles or to tissue-specific genes. All expression clusters are enriched in effector genes, and one cluster is specific to the saprophytic lifestyle on plant residues. One cluster, including genes known to be involved in the first phase of asymptomatic fungal growth in leaves, is re-used at each asymptomatic growth stage, regardless of the type of organ infected. The expression of the genes of this cluster is repeatedly turned on and off during infection. Whatever their expression profile, the genes of these clusters are enriched in heterochromatin regions associated with H3K9me3 or H3K27me3 repressive marks. These findings provide support for the hypothesis that part of the fungal genes involved in niche adaptation is located in heterochromatic regions of the genome, conferring an extreme plasticity of expression. Conclusion This work opens up new avenues for plant disease control, by identifying stage-specific effectors that could be used as targets for the identification of novel durable disease resistance genes, or for the in-depth analysis of chromatin remodeling during plant infection, which could be manipulated to interfere with the global expression of effector genes at crucial stages of plant infection.

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Differential Expression of BARD1 Isoforms in Melanoma

Genes ◽

10.3390/genes12020320 ◽

2021 ◽

Vol 12 (2) ◽

pp. 320

Author(s):

Lorissa I. McDougall ◽

Ryan M. Powell ◽

Magdalena Ratajska ◽

Chi F. Lynch-Sutherland ◽

Sultana Mehbuba Hossain ◽

...

Keyword(s):

Long Range ◽

Patient Outcomes ◽

Splice Variants ◽

Significant Proportion ◽

Nanopore Sequencing ◽

Rna Seq ◽

Splice Isoforms ◽

Tissue Samples ◽

Multiple Transcripts ◽

Mrna Variants

Melanoma comprises <5% of cutaneous malignancies, yet it causes a significant proportion of skin cancer-related deaths worldwide. While new therapies for melanoma have been developed, not all patients respond well. Thus, further research is required to better predict patient outcomes. Using long-range nanopore sequencing, RT-qPCR, and RNA sequencing analyses, we examined the transcription of BARD1 splice isoforms in melanoma cell lines and patient tissue samples. Seventy-six BARD1 mRNA variants were identified in total, with several previously characterised isoforms (γ, φ, δ, ε, and η) contributing to a large proportion of the expressed transcripts. In addition, we identified four novel splice events, namely, Δ(E3_E9), ▼(i8), IVS10+131▼46, and IVS10▼176, occurring in various combinations in multiple transcripts. We found that short-read RNA-Seq analyses were limited in their ability to predict isoforms containing multiple non-contiguous splicing events, as compared to long-range nanopore sequencing. These studies suggest that further investigations into the functional significance of the identified BARD1 splice variants in melanoma are warranted.

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