scholarly journals Low-cost, low-input RNA-seq protocols perform nearly as well as high-input protocols

2015 ◽  
Author(s):  
Peter A Combs ◽  
Michael B Eisen
Keyword(s):  
Low Cost ◽  
Critical Evaluation ◽  
Standard Protocol ◽  
Library Preparation ◽  
Rna Seq ◽  
High Input ◽  
Simple Modification ◽  
Input Sample ◽  
Low Volume ◽  
The Cost

Recently, a number of protocols extending RNA-sequencing to the single-cell regime have been published. However, we were concerned that the additional steps to deal with such minute quantities of input sample would introduce serious biases that would make analysis of the data using existing approaches invalid. In this study, we performed a critical evaluation of several of these low-volume RNA-seq protocols, and found that they performed slightly less well in metrics of interest to us than a more standard protocol, but with at least two orders of magnitude less sample required. We also explored a simple modification to one of these protocols that, for many samples, reduced the cost of library preparation to approximately $20/sample.

scholarly journals Low-cost, low-input RNA-seq protocols perform nearly as well as high-input protocols

2015 ◽  
Author(s):  
Peter A Combs ◽  
Michael B Eisen
Keyword(s):  
Low Cost ◽  
Critical Evaluation ◽  
Standard Protocol ◽  
Library Preparation ◽  
Rna Seq ◽  
High Input ◽  
Simple Modification ◽  
Input Sample ◽  
Low Volume ◽  
The Cost

Recently, a number of protocols extending RNA-sequencing to the single-cell regime have been published. However, we were concerned that the additional steps to deal with such minute quantities of input sample would introduce serious biases that would make analysis of the data using existing approaches invalid. In this study, we performed a critical evaluation of several of these low-volume RNA-seq protocols, and found that they performed slightly less well in metrics of interest to us than a more standard protocol, but with at least two orders of magnitude less sample required. We also explored a simple modification to one of these protocols that, for many samples, reduced the cost of library preparation to approximately $20/sample.

scholarly journals Validation of methods for Low-volume RNA-seq

2014 ◽  
Author(s):  
Peter Acuña Combs ◽  
Michael B Eisen
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Critical Evaluation ◽  
Standard Protocol ◽  
Library Preparation ◽  
Rna Seq ◽  
Simple Modification ◽  
Input Sample ◽  
Low Volume ◽  
The Cost

Recently, a number of protocols extending RNA-sequencing to the single-cell regime have been published. However, we were concerned that the additional steps to deal with such minute quantities of input sample would introduce serious biases that would make analysis of the data using existing approaches invalid. In this study, we performed a critical evaluation of several of these low-volume RNA-seq protocols, and found that they performed slightly less well in metrics of interest to us than a more standard protocol, but with at least two orders of magnitude less sample required. We also explored a simple modification to one of these protocols that, for many samples, reduced the cost of library preparation to approximately $20/sample.

scholarly journals TM3’seq: A Tagmentation-Mediated 3’ Sequencing Approach for Improving Scalability of RNAseq Experiments

2019 ◽  
Vol 10 (1) ◽  
pp. 143-150 ◽  
Author(s):  
Luisa F. Pallares ◽  
Serge Picard ◽  
Julien F. Ayroles
Keyword(s):  
Single Cell ◽  
Large Scale ◽  
Enriched Library ◽  
Library Preparation ◽  
Rna Seq ◽  
Medical Genomics ◽  
Genome Wide ◽  
Standard Tool ◽  
Commercial Kits ◽  
The Cost

RNA-seq has become the standard tool for collecting genome-wide expression data in diverse fields, from quantitative genetics and medical genomics to ecology and developmental biology. However, RNA-seq library preparation is still prohibitive for many laboratories. Recently, the field of single-cell transcriptomics has reduced costs and increased throughput by adopting early barcoding and pooling of individual samples —producing a single final library containing all samples. In contrast, RNA-seq protocols where each sample is processed individually are significantly more expensive and lower throughput than single-cell approaches. Yet, many projects depend on individual library generation to preserve important samples or for follow-up re-sequencing experiments. Improving on currently available RNA-seq methods we have developed TM3′seq, a 3′-enriched library preparation protocol that uses Tn5 transposase and preserves sample identity at each step. TM3′seq is designed for high-throughput processing of individual samples (96 samples in 6h, with only 3h hands-on time) at a fraction of the cost of commercial kits ($1.5 per sample). The protocol was tested in a range of human and Drosophila melanogaster RNA samples, recovering transcriptomes of the same quality and reliability than the commercial NEBNext kit. We expect that the cost- and time-efficient features of TM3′seq make large-scale RNA-seq experiments more permissive for the entire scientific community.

scholarly journals Low-cost and High-throughput RNA-seq Library Preparation for Illumina Sequencing from Plant Tissue

BIO-PROTOCOL ◽  
2020 ◽  
Vol 10 (20) ◽  
Author(s):  
Marta Bjornson ◽  
Kaisa Kajala ◽  
Cyril Zipfel ◽  
Pingtao Ding
Keyword(s):  
High Throughput ◽  
Illumina Sequencing ◽  
Plant Tissue ◽  
Low Cost ◽  
Library Preparation ◽  
Rna Seq

scholarly journals Time- and cost-efficient high-throughput transcriptomics enabled by Bulk RNA Barcoding and sequencing

2018 ◽  
Author(s):  
Daniel Alpern ◽  
Vincent Gardeux ◽  
Julie Russeil ◽  
Bart Deplancke
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
High Throughput ◽  
Low Cost ◽  
Cdna Libraries ◽  
List Type ◽  
Library Preparation ◽  
Rna Seq ◽  
Genome Wide ◽  
Hands On

ABSTRACTGenome-wide gene expression analyses by RNA sequencing (RNA-seq) have quickly become a standard in molecular biology because of the widespread availability of high throughput sequencing technologies. While powerful, RNA-seq still has several limitations, including the time and cost of library preparation, which makes it difficult to profile many samples simultaneously. To deal with these constraints, the single-cell transcriptomics field has implemented the early multiplexing principle, making the library preparation of hundreds of samples (cells) markedly more affordable. However, the current standard methods for bulk transcriptomics (such as TruSeq Stranded mRNA) remain expensive, and relatively little effort has been invested to develop cheaper, but equally robust methods. Here, we present a novel approach, Bulk RNA Barcoding and sequencing (BRB-seq), that combines the multiplexing-driven cost-effectiveness of a single-cell RNA-seq workflow with the performance of a bulk RNA-seq procedure. BRB-seq produces 3’ enriched cDNA libraries that exhibit similar gene expression quantification to TruSeq and that maintain this quality, also in terms of number of detected differentially expressed genes, even with low quality RNA samples. We show that BRB-seq is about 25 times less expensive than TruSeq, enabling the generation of ready to sequence libraries for up to 192 samples in a day with only 2 hours of hands-on time. We conclude that BRB-seq constitutes a powerful alternative to TruSeq as a standard bulk RNA-seq approach. Moreover, we anticipate that this novel method will eventually replace RT-qPCR-based gene expression screens given its capacity to generate genome-wide transcriptomic data at a cost that is comparable to profiling 4 genes using RT-qPCR.‘SoftwareWe developed a suite of open source tools (BRB-seqTools) to aid with processing BRB-seq data and generating count matrices that are used for further analyses. This suite can perform demultiplexing, generate count/UMI matrices and trim BRB-seq constructs and is freely available at http://github.com/DeplanckeLab/BRB-seqToolsHighlightsRapid (~2h hands on time) and low-cost approach to perform transcriptomics on hundreds of RNA samplesStrand specificity preservedPerformance: number of detected genes is equal to Illumina TruSeq Stranded mRNA at same sequencing depthHigh capacity: low cost allows increasing the number of biological replicatesProduces reliable data even with low quality RNA samples (down to RIN value = 2)Complete user-friendly sequencing data pre-processing and analysis pipeline allowing result acquisition in a day

scholarly journals Lasy-Seq: a high-throughput library preparation method for RNA-Seq and its application in the analysis of plant responses to fluctuating temperatures

2018 ◽  
Author(s):  
Mari Kamitani ◽  
Makoto Kashima ◽  
Ayumi Tezuka ◽  
Atsushi J. Nagano
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Preparation Method ◽  
Low Cost ◽  
Cost Effective ◽  
Plant Responses ◽  
Library Preparation ◽  
Rna Seq ◽  
Temperature Responses ◽  
Library Preparation Method

AbstractRNA-Seq is a whole-transcriptome analysis method used to research biological mechanisms and functions; its use in large-scale experiments is limited by costs and labour. In this study, we established a high-throughput and cost effective RNA-Seq library preparation method that did not require mRNA enrichment. The method adds unique index sequences to samples during reverse transcription (RT) that is conducted at a higher temperature (≥62°C) to suppress RT of A-rich sequences in rRNA, and then pools all samples into a single tube. Both single-read and paired end sequencing of libraries is enabled. We found that the pooled RT products contained large amounts of RNA, mainly rRNA, and caused over-estimations of the quantity of DNA, resulting in unstable tagmentation results. Degradation of RNA before tagmentation was necessary for the stable preparation of libraries. We named this protocol low-cost and easy RNA-Seq (Lasy-Seq), and used it to investigate temperature responses in Arabidopsis thaliana. We analysed how sub-ambient temperatures (10–30°C) affected the plant transcriptomes, using time-courses of RNA-Seq from plants grown in randomly fluctuating temperature conditions. Our results suggest that there are diverse mechanisms behind plant temperature responses at different time scales.

scholarly journals TM3’seq: a tagmentation-mediated 3’ sequencing approach for improving scalability of RNA-seq experiments

2019 ◽  
Author(s):  
Luisa F. Pallares ◽  
Serge Picard ◽  
Julien F. Ayroles
Keyword(s):  
Single Cell ◽  
Large Scale ◽  
Enriched Library ◽  
Library Preparation ◽  
Rna Seq ◽  
Medical Genomics ◽  
Genome Wide ◽  
Standard Tool ◽  
Commercial Kits ◽  
The Cost

AbstractRNA-seq has become the standard tool for collecting genome-wide expression data in diverse fields, from quantitative genetics and medical genomics to ecology and developmental biology. However, RNA-seq library preparation is still prohibitive for many laboratories. Recently, the field of single-cell transcriptomics has reduced costs and increased throughput by adopting early barcoding and pooling of individual samples —producing a single final library containing all samples. In contrast, RNA-seq protocols where each sample is processed individually are significantly more expensive and lower throughput than single-cell approaches. Yet, many projects depend on individual library generation to preserve important samples or for follow-up re-sequencing experiments. Improving on currently available RNA-seq methods we have developed TM3’seq, a 3’-enriched library preparation protocol that uses Tn5 transposase and preserves sample identity at each step. TM3’seq is designed for high-throughput processing of individual samples (96 samples in 6h, with only 3h hands-on time) at a fraction of the cost of commercial kits ($1.5 per sample). The protocol was tested in a range of human andDrosophila melanogasterRNA samples, recovering transcriptomes of the same quality and reliability than the commercial NEBNext®kit. We expect that the cost-and time-efficient features of TM3’seq make large-scale RNA-seq experiments more permissive for the entire scientific community.

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