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

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Mari Kamitani ◽  
Makoto Kashima ◽  
Ayumi Tezuka ◽  
Atsushi J. Nagano
Keyword(s):  
High Throughput ◽  
Preparation Method ◽  
Plant Responses ◽  
Library Preparation ◽  
Rna Seq ◽  
Fluctuating Temperatures ◽  
Library Preparation Method

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.

Transcriptome-wide m6A detection with DART-Seq

2019 ◽  
Author(s):  
Kate D. Meyer
Keyword(s):  
Gene Expression Regulation ◽  
Preparation Method ◽  
Current Knowledge ◽  
Cross Reactivity ◽  
High Variability ◽  
Library Preparation ◽  
Rna Seq ◽  
Generation Sequencing ◽  
Library Preparation Method

Abstract m6A is the most abundant internal mRNA modification and plays diverse roles in gene expression regulation. Much of our current knowledge about m6A has been driven by recent advances in the ability to detect this mark transcriptome-wide. Antibody-based approaches have been the method of choice for global m6A mapping studies. These methods rely on m6A antibodies to immunoprecipitate methylated RNAs, followed by next-generation sequencing to identify m6A-containing transcripts1,2. While these methods enabled the first identification of m6A sites transcriptome-wide and have dramatically improved our ability to study m6A, they suffer from several limitations. These include requirements for high amounts of input RNA, costly and time-consuming library preparation, high variability across studies, and m6A antibody cross-reactivity with other modifications. Here, we describe DART-Seq (deamination adjacent to RNA modification targets), an antibody-free method for global m6A detection. In DART-Seq, the C to U deaminating enzyme, APOBEC1, is fused to the m6A-binding YTH domain. This fusion protein is then introduced to cellular RNA either through overexpression in cells or with in vitro assays, and subsequent deamination of m6A-adjacent cytidines is then detected by RNA sequencing to identify m6A sites. DART-Seq can successfully map m6A sites throughout the transcriptome using as little as 10 nanograms of total cellular RNA, and it is compatible with any standard RNA-seq library preparation method.

scholarly journals Comparison of Poly-A+ Selection and rRNA Depletion in Detection of lncRNA in Two Equine Tissues Using RNA-seq

2020 ◽  
Vol 6 (3) ◽  
pp. 32 ◽  
Author(s):  
Anna R. Dahlgren ◽  
Erica Y. Scott ◽  
Tamer Mansour ◽  
Erin N. Hales ◽  
Pablo J. Ross ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Preparation Method ◽  
Parietal Lobe ◽  
Library Preparation ◽  
Rna Seq ◽  
The Past ◽  
Rrna Depletion ◽  
Non Coding Rnas ◽  
Nucleolar Rna ◽  
Library Preparation Method

Long non-coding RNAs (lncRNAs) are untranslated regulatory transcripts longer than 200 nucleotides that can play a role in transcriptional, post-translational, and epigenetic regulation. Traditionally, RNA-sequencing (RNA-seq) libraries have been created by isolating transcriptomic RNA via poly-A+ selection. In the past 10 years, methods to perform ribosomal RNA (rRNA) depletion of total RNA have been developed as an alternative, aiming for better coverage of whole transcriptomic RNA, both polyadenylated and non-polyadenylated transcripts. The purpose of this study was to determine which library preparation method is optimal for lncRNA investigations in the horse. Using liver and cerebral parietal lobe tissues from two healthy Thoroughbred mares, RNA-seq libraries were prepared using standard poly-A+ selection and rRNA-depletion methods. Averaging the two biologic replicates, poly-A+ selection yielded 327 and 773 more unique lncRNA transcripts for liver and parietal lobe, respectively. More lncRNA were found to be unique to poly-A+ selected libraries, and rRNA-depletion identified small nucleolar RNA (snoRNA) to have a higher relative expression than in the poly-A+ selected libraries. Overall, poly-A+ selection provides a more thorough identification of total lncRNA in equine tissues while rRNA-depletion may allow for easier detection of snoRNAs.

scholarly journals High throughput barcoding method for genome-scale phasing

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
David Redin ◽  
Tobias Frick ◽  
Hooman Aghelpasand ◽  
Max Käller ◽  
Erik Borgström ◽  
...  
Keyword(s):  
High Throughput ◽  
Preparation Method ◽  
Library Preparation ◽  
Structural Variants ◽  
Human Genomics ◽  
Short Read Sequencing ◽  
Base Calling ◽  
Cost Efficient ◽  
Genome Scale ◽  
Library Preparation Method

AbstractThe future of human genomics is one that seeks to resolve the entirety of genetic variation through sequencing. The prospect of utilizing genomics for medical purposes require cost-efficient and accurate base calling, long-range haplotyping capability, and reliable calling of structural variants. Short-read sequencing has lead the development towards such a future but has struggled to meet the latter two of these needs. To address this limitation, we developed a technology that preserves the molecular origin of short sequencing reads, with an insignificant increase to sequencing costs. We demonstrate a novel library preparation method for high throughput barcoding of short reads where millions of random barcodes can be used to reconstruct megabase-scale phase blocks.

scholarly journals High-Throughput Miniaturized 16S rRNA Amplicon Library Preparation Reduces Costs while Preserving Microbiome Integrity

mSystems ◽  
2018 ◽  
Vol 3 (6) ◽  
Author(s):  
Jeremiah J. Minich ◽  
Greg Humphrey ◽  
Rodolfo A. S. Benitez ◽  
Jon Sanders ◽  
Austin Swafford ◽  
...  
Keyword(s):  
16S Rrna ◽  
Microbial Ecology ◽  
High Throughput ◽  
Preparation Method ◽  
Library Preparation ◽  
Reaction Volume ◽  
Liquid Handling ◽  
Amplicon Library ◽  
The Cost ◽  
Library Preparation Method

ABSTRACT 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 (R2 > 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.

scholarly journals Mosaic Ends Tagmentation (METa) assembly for extremely efficient construction of functional metagenomic libraries

2021 ◽  
Author(s):  
Terence S. Crofts ◽  
Alexander G. McFarland ◽  
Erica M. Hartmann
Keyword(s):  
Amino Acid ◽  
High Throughput ◽  
Preparation Method ◽  
Metagenomic Library ◽  
Amino Acid Identity ◽  
Library Preparation ◽  
Novel Genes ◽  
Acid Identity ◽  
Metagenomic Libraries ◽  
Library Preparation Method

ABSTRACTFunctional metagenomic libraries, physical bacterial libraries which allow the high-throughput capture and expression of microbiome genes, have been instrumental in the sequence-naïve and cultivation-independent discovery of novel genes from microbial communities. Preparation of these libraries is limited by their high DNA input requirement and their low cloning efficiency. Here, we describe a new method, METa assembly, for extremely efficient functional metagenomic library preparation. We apply tagmentation to metagenomic DNA from soil and gut microbiomes to prepare DNA inserts for high-throughput cloning into functional metagenomic libraries. The presence of mosaic end sequences in the resulting DNA fragments synergizes with homology-based assembly cloning to result in a 300-fold increase in library size compared to traditional blunt cloning based protocols. Compared to published libraries prepared by state-of-the-art protocols we show that METa assembly is on average 23- to 270-fold more efficient and can be effectively used to prepare gigabase-sized libraries with as little as 200 ng of input DNA. We demonstrate the utility of METa assembly to capture novel genes based on their function by discovering novel aminoglycoside (26% amino acid identity) and colistin (36% amino acid identity) resistance genes in soil and goose gut microbiomes. METa assembly provides a streamlined, flexible, and efficient method for preparing functional metagenomic libraries, enabling new avenues of genetic and biochemical research into low biomass or scarce microbiomes.IMPORTANCEMedically and industrially important genes can be recovered from microbial communities by high-throughput sequencing but are limited to previously sequenced genes and their relatives. Cloning a metagenome en masse into an expression host to produce a functional metagenomic library is a sequence-naïve and cultivation-independent method to discover novel genes. This directly connects genes to functions, but the process of preparing these libraries is DNA greedy and inefficient. Here we describe a library preparation method that is an order of magnitude more efficient and less DNA greedy. This method is consistently efficient across libraries prepared from cultures, a soil microbiome, and from a goose fecal microbiome and allowed us to discover novel antibiotic resistance genes. This new library preparation method will potentially allow for the functional metagenomic exploration of microbiomes that were previously off limits due to their rarity or low microbial biomass, such biomedical swabs or exotic samples.

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