High-throughput Minitaturized RNA-Seq Library Preparation.
Ontology highlight
ABSTRACT:
SUBMITTER: Mildrum S
PROVIDER: S-EPMC7566612 | biostudies-literature | 2020 Dec
REPOSITORIES: biostudies-literature
Publications
High-throughput Minitaturized RNA-Seq Library Preparation.
Mildrum Samuel S Hendricks Austin A Stortchevoi Alexei A Kamelamela Noelani N Butty Vincent L VL Levine Stuart S SS
Journal of biomolecular techniques : JBT 20201201 4
Advances in next-generation sequencing technologies have allowed RNA sequencing to become an increasingly time efficient, cost-effective, and accessible tool for genomic research. We present here an automated and miniaturized workflow for RNA library preparation that minimizes reagent usage and processing time required per sample to generate Illumina compatible libraries for sequencing. The reduced-volume libraries show similar behavior to full-scale libraries with comparable numbers of genes de ...[more]
Similar Datasets
Project description:With the introduction of cost effective, rapid, and superior quality next generation sequencing techniques, gene expression analysis has become viable for labs conducting small projects as well as large-scale gene expression analysis experiments. However, the available protocols for construction of RNA-sequencing (RNA-Seq) libraries are expensive and/or difficult to scale for high-throughput applications. Also, most protocols require isolated total RNA as a starting point. We provide a cost-effective RNA-Seq library synthesis protocol that is fast, starts with tissue, and is high-throughput from tissue to synthesized library. We have also designed and report a set of 96 unique barcodes for library adapters that are amenable to high-throughput sequencing by a large combination of multiplexing strategies. Our developed protocol has more power to detect differentially expressed genes when compared to the standard Illumina protocol, probably owing to less technical variation amongst replicates. We also address the problem of gene-length biases affecting differential gene expression calls and demonstrate that such biases can be efficiently minimized during mRNA isolation for library preparation.
| S-EPMC3428589 | biostudies-literature
Project description:RNA-Seq is a whole-transcriptome analysis method used to research biological mechanisms and functions but its use in large-scale experiments is limited by its high cost and labour requirements. In this study, we have established a high-throughput and cost-effective RNA-Seq library preparation method that does 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, causing over-estimations of the quantity of DNA and unstable tagmentation results. Degradation of RNA before tagmentation was found to be 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.
| S-EPMC6506593 | biostudies-literature
Project description:BACKGROUND:Current library preparation protocols for Illumina HiSeq and MiSeq DNA sequencers require ?2 nM initial library for subsequent loading of denatured cDNA onto flow cells. Such amounts are not always attainable from samples having a relatively low DNA or RNA input; or those for which a limited number of PCR amplification cycles is preferred (less PCR bias and/or more even coverage). A well-tested sub-nanomolar library preparation protocol for Illumina sequencers has however not been reported. The aim of this study is to provide a much needed working protocol for sub-nanomolar libraries to achieve outcomes as informative as those obtained with the higher library input (? 2 nM) recommended by Illumina's protocols. RESULTS:Extensive studies were conducted to validate a robust sub-nanomolar (initial library of 100 pM) protocol using PhiX DNA (as a control), genomic DNA (Bordetella bronchiseptica and microbial mock community B for 16S rRNA gene sequencing), messenger RNA, microRNA, and other small noncoding RNA samples. The utility of our protocol was further explored for PhiX library concentrations as low as 25 pM, which generated only slightly fewer than 50% of the reads achieved under the standard Illumina protocol starting with >?2 nM. CONCLUSIONS:A sub-nanomolar library preparation protocol (100 pM) could generate next generation sequencing (NGS) results as robust as the standard Illumina protocol. Following the sub-nanomolar protocol, libraries with initial concentrations as low as 25 pM could also be sequenced to yield satisfactory and reproducible sequencing results.
| S-EPMC5935984 | biostudies-literature
Project description:Library preparation for high-throughput sequencing applications is a critical step in producing representative, unbiased sequencing data. The iGenomX Riptide High Throughput Rapid Library Prep Kit purports to provide high-quality sequencing data with lower costs compared to other Illumina library kits. To test these claims, we compared sequence data quality of Riptide libraries to libraries constructed with KAPA Hyper and NEBNext Ultra. Across several single-source genome samples, mapping performance and de novo assembly of Riptide libraries were similar to conventional libraries prepared with the same DNA. Poor performance of some libraries resulted in low sequencing depth. In particular, degraded DNA samples may be challenging to sequence with Riptide. There was little cross-well plate contamination with the overwhelming majority of reads belong to the proper source genomes. The sequencing of metagenome samples using different Riptide primer sets resulted in variable taxonomic assignment of reads. Increased adoption of the Riptide kit will decrease library preparation costs. However, this method might not be suitable for degraded DNA.
| S-EPMC8342411 | biostudies-literature
Project description:BackgroundmRNA sequencing is a powerful technique, which is used to investigate the transcriptome status of a gene of interest, such as its transcription level and splicing variants. Presently, several RNA sequencing (RNA-Seq) methods have been developed; however, the relative advantage of each method has remained unknown. Here we used three commercially available RNA-Seq library preparation kits; the traditional method (TruSeq), in addition to full-length double-stranded cDNA methods (SMARTer and TeloPrime) to investigate the advantages and disadvantages of these three approaches in transcriptome analysis.ResultsWe observed that the number of expressed genes detected from the TeloPrime sequencing method was fewer than that obtained using the TruSeq and SMARTer. We also observed that the expression patterns between TruSeq and SMARTer correlated strongly. Alternatively, SMARTer and TeloPrime methods underestimated the expression of relatively long transcripts. Moreover, genes having low expression levels were undetected stochastically regardless of any three methods used. Furthermore, although TeloPrime detected a significantly higher proportion at the transcription start site (TSS), its coverage of the gene body was not uniform. SMARTer is proposed to be yielded for nonspecific genomic DNA amplification. In contrast, the detected splicing event number was highest in the TruSeq. The percent spliced in index (PSI) of the three methods was highly correlated.ConclusionsTruSeq detected transcripts and splicing events better than the other methods and measured expression levels of genes, in addition to splicing events accurately. However, although detected transcripts and splicing events in TeloPrime were fewer, the coverage at TSS was highest. Additionally, SMARTer was better than TeloPrime with regards to the detected number of transcripts and splicing events among the understudied full-length double-stranded cDNA methods. In conclusion, for short-read sequencing, TruSeq has relative advantages for use in transcriptome analysis.
| S-EPMC9008973 | biostudies-literature
Project description:Library preparation is a key step in sequencing. For RNA sequencing there are advantages to both strand specificity and working with minute starting material, yet until recently there was no kit available enabling both. The Illumina TruSeq stranded mRNA Sample Preparation kit (TruSeq) requires abundant starting material while the Takara Bio SMART-Seq v4 Ultra Low Input RNA kit (V4) sacrifices strand specificity. The SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Pico) by Takara Bio claims to overcome these limitations. Comparative evaluation of these kits is important for selecting the appropriate protocol. We compared the three kits in a realistic differential expression analysis. We prepared and sequenced samples from two experimental conditions of biological interest with each of the three kits. We report differences between the kits at the level of differential gene expression; for example, the Pico kit results in 55% fewer differentially expressed genes than TruSeq. Nevertheless, the agreement of the observed enriched pathways suggests that comparable functional results can be obtained. In summary we conclude that the Pico kit sufficiently reproduces the results of the other kits at the level of pathway analysis while providing a combination of options that is not available in the other kits.
| S-EPMC6748930 | biostudies-literature
Project description:The Nextera protocol, which utilises a transposome based approach to create libraries for Illumina sequencing, requires pure DNA template, an accurate assessment of input concentration and a column clean-up that limits its applicability for high-throughput sample preparation. We addressed the identified limitations to develop a robust workflow that supports both rapid and high-throughput projects also reducing reagent costs.We show that an initial bead-based normalisation step can remove the need for quantification and improves sample purity. A 75% cost reduction was achieved with a low-volume modified protocol which was tested over genomes with different GC content to demonstrate its robustness. Finally we developed a custom set of index tags and primers which increase the number of samples that can simultaneously be sequenced on a single lane of an Illumina instrument.We addressed the bottlenecks of Nextera library construction to produce a modified protocol which harnesses the full power of the Nextera kit and allows the reproducible construction of libraries on a high-throughput scale reducing the associated cost of the kit.
| S-EPMC4222894 | biostudies-literature
Project description:BackgroundSingle-cell RNA sequencing (scRNA-seq) provides new insights to address biological and medical questions, and it will benefit more from the ultralow input RNA or subcellular sequencing.ResultsHere, we present a highly sensitive library construction protocol for ultralow input RNA sequencing (ulRNA-seq). We systematically evaluate experimental conditions of this protocol, such as reverse transcriptase, template-switching oligos (TSO), and template RNA structure. It was found that Maxima H Minus reverse transcriptase and rN modified TSO, as well as all RNA templates capped with m7G improved the sequencing sensitivity and low abundance gene detection ability. RNA-seq libraries were successfully prepared from total RNA samples as low as 0.5 pg, and more than 2000 genes have been identified.ConclusionsThe ability of low abundance gene detection and sensitivity were largely enhanced with this optimized protocol. It was also confirmed in single-cell sequencing, that more genes and cell markers were identified compared to conventional sequencing method. We expect that ulRNA-seq will sequence and transcriptome characterization for the subcellular of disease tissue, to find the corresponding treatment plan.
| S-EPMC8579666 | biostudies-literature
Project description:High-throughput sequencing (HTS) of antibody repertoire libraries has become a powerful tool in the field of systems immunology. However, numerous sources of bias in HTS workflows may affect the obtained antibody repertoire data. A crucial step in antibody library preparation is the addition of short platform-specific nucleotide adapter sequences. As of yet, the impact of the method of adapter addition on experimental library preparation and the resulting antibody repertoire HTS datasets has not been thoroughly investigated. Therefore, we compared three standard library preparation methods by performing Illumina HTS on antibody variable heavy genes from murine antibody-secreting cells. Clonal overlap and rank statistics demonstrated that the investigated methods produced equivalent HTS datasets. PCR-based methods were experimentally superior to ligation with respect to speed, efficiency, and practicality. Finally, using a two-step PCR based method we established a protocol for antibody repertoire library generation, beginning from inputs as low as 1 ng of total RNA. In summary, this study represents a major advance towards a standardized experimental framework for antibody HTS, thus opening up the potential for systems-based, cross-experiment meta-analyses of antibody repertoires.
| S-EPMC4014543 | biostudies-literature
Project description:Next-generation sequencing technologies have enabled many advances across biology, with microbial ecology benefiting primarily through expanded sample sizes. Although the cost of running sequencing instruments has decreased substantially over time, the price of library preparation methods has largely remained unchanged. In this study, we developed a low-cost miniaturized (5-µl volume) high-throughput (384-sample) amplicon library preparation method with the Echo 550 acoustic liquid handler. Our method reduces costs of library preparation to $1.42 per sample, a 58% reduction compared to existing automated methods and a 21-fold reduction from commercial kits, without compromising sequencing success or distorting the microbial community composition analysis. We further validated the optimized method by sampling five body sites from 46 Pacific chub mackerel fish caught across 16 sampling events over seven months from the Scripps Institution of Oceanography pier in La Jolla, CA. Fish microbiome samples were processed with the miniaturized 5-µl reaction volume with 0.2 µl of genomic DNA (gDNA) and the standard 25-µl reaction volume with 1 µl of gDNA. Between the two methods, alpha diversity was highly correlated (R 2 > 0.95), while distances of technical replicates were much lower than within-body-site variation (P < 0.0001), further validating the method. The cost savings of implementing the miniaturized library preparation (going from triplicate 25-µl reactions to triplicate 5-µl reactions) are large enough to cover a MiSeq sequencing run for 768 samples while preserving accurate microbiome measurements. IMPORTANCE Reduced costs of sequencing have tremendously impacted the field of microbial ecology, allowing scientists to design more studies with larger sample sizes that often exceed 10,000 samples. Library preparation costs have not kept pace with sequencing prices, although automated liquid handling robots provide a unique opportunity to bridge this gap while also decreasing human error. Here, we take advantage of an acoustic liquid handling robot to develop a high-throughput miniaturized library preparation method of a highly cited and broadly used 16S rRNA gene amplicon reaction. We evaluate the potential negative effects of reducing the PCR volume along with varying the amount of gDNA going into the reaction. Our optimized method reduces sample-processing costs while continuing to generate a high-quality microbiome readout that is indistinguishable from the original method.
| S-EPMC6222042 | biostudies-literature