External RNA Controls Consortium Beta Version Update

Lee, Hangnoh; Pine, P. Scott; McDaniel, Jennifer; Salit, Marc; Oliver, Brian

doi:10.7150/jgen.16082

Cited by 32 publications

(36 citation statements)

References 7 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Paired-end sequencing with a read length of 100 bp was carried out on an Illumina HiSeq 2000 at the Genomic Sequencing Unit of the University of Dundee. ERCC RNA Spike-In mixes (Thermo Fisher Scientific) 11, 51 were included in each of the libraries using concentrations advised by the manufacturer.…”

Section: Methodsmentioning

confidence: 99%

“…Reads were basecalled with Guppy version 2.3.1 (Oxford Nanopore Technologies) using default RNA parameters and converted from RNA to DNA fastq using seqkit version 0.10.0 59 . Reads were aligned to the TAIR10 A. thaliana genome 60 and ERCC RNA Spike-In sequences 11, 51 using minimap2 version 2.8 13 in spliced mapping mode using a kmer size of 14 and a maximum intron size of 10,000 nt. Sequence Alignment/Map (SAM) and BAM file manipulations were performed using samtools version 1.9 56 .…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Nanopore direct RNA sequencing maps an Arabidopsis N6 methyladenosine epitranscriptome

Parker

Knop

Sherwood

et al. 2019

Preprint

View full text Add to dashboard Cite

21Understanding genome organization and gene regulation requires insight into RNA transcription, 22 processing and modification. We adapted nanopore direct RNA sequencing to examine RNA from a 23 wild-type accession of the model plant Arabidopsis thaliana and a mutant defective in mRNA 24 methylation (m 6 A). Here we show that m 6 A can be mapped in full-length mRNAs transcriptome-wide 25 and reveal the combinatorial diversity of cap-associated transcription start sites, splicing events, 26 poly(A) site choice and poly(A) tail length. Loss of m 6 A from 3' untranslated regions is associated 27 with decreased relative transcript abundance and defective RNA 3′ end formation. A functional 28 consequence of disrupted m 6 A is a lengthening of the circadian period. We conclude that nanopore 29 direct RNA sequencing can reveal the complexity of mRNA processing and modification in full-length 30 single molecule reads. These findings can refine Arabidopsis genome annotation. Further, applying 31 this approach to less well-studied species could transform our understanding of what their genomes 32 encode. 33 34 misidentification of 3′ ends through internal priming 3 , spurious antisense and splicing events 46 produced by RT template switching 4,5 , and the inability to detect all base modifications in the 47 copying process 6 . The fragmentation of RNA prior to short-read sequencing makes it difficult to 48 interpret the combination of authentic RNA processing events and remains an unsolved problem 7 . 49We investigated whether long-read direct RNA sequencing (DRS) with nanopores 8 could 50 reveal the complexity of Arabidopsis mRNA processing and modifications. In nanopore DRS, the 51 protein pore (nanopore) sits in a membrane through which an electrical current is passed, and intact 52 RNA is fed through the nanopore by a motor protein 8 . Each RNA sequence within the nanopore 53 (5 bases) can be identified by the magnitude of signal it produces. Arabidopsis is a pathfinder model 54 in plant biology, and its genome annotation strongly influences the annotation and our 55 understanding of what other plant genomes encode. We applied nanopore DRS and Illumina RNAseq 56 to wild-type Arabidopsis (Col-0) and mutants defective in m 6 A 9 and exosome-mediated RNA decay 10 . 57We reveal m 6 A and combinations of RNA processing events (alternative patterns of 5′ capped 58 transcription start sites, splicing, 3′ polyadenylation and poly(A) tail length) in full-length Arabidopsis 59 mRNAs transcriptome-wide. 60 61 Results 62Nanopore DRS detects long, complex mRNAs and short, structured non-coding RNAs 63We purified poly (A)+ RNA from four biological replicates of 14-day-old Arabidopsis Col-0 seedlings. 64We incorporated synthetic External RNA Controls Consortium (ERCC) RNA Spike-In mixes into all 65 replicates 11,12 and carried out nanopore DRS. Illumina RNAseq was performed in parallel on similar 66 material. Using Guppy base-calling (Oxford Nanopore Technologies) and minimap2 alignment 67 software 13 , we identified around 1 mi...

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Nanopore direct RNA sequencing maps an Arabidopsis N6 methyladenosine epitranscriptome

Parker

Knop

Sherwood

et al. 2019

Preprint

View full text Add to dashboard Cite

show abstract

“…Internal spike-in control nucleic acids are useful indicators of potential library preparation errors. Furthermore, carefully designed spike-in controls, such as the External RNA Controls Consortium (ERCC) collection [10–12], which consists of 92 variable-length archaeal templates present at a pre-defined range of concentrations, may be used to establish the relationship between read count and input RNA concentration. For this mNGS protocol, 25 picograms of ERCC RNA (Thermo Fisher Scientific Cat.…”

Section: Methods and Resultsmentioning

confidence: 99%

Miniaturization and optimization of 384-well compatible metagenomic sequencing library preparation

et al. 2018

Preprint

View full text Add to dashboard Cite

17 Preparation of high-quality sequencing libraries is a costly and time-consuming component of 18 metagenomic next generation sequencing (mNGS). While the overall cost of sequencing has 19 dropped significantly over recent years, the reagents needed to prepare sequencing samples are 20 likely to become the dominant expense in the process. Furthermore, libraries prepared by hand 21 are subject to human variability and needless waste due to limitations of manual pipetting 22 volumes. Reduction of reaction volumes, combined with sub-microliter automated dispensing of 23 reagents without consumable pipette tips, has the potential to provide significant advantages.24 Here, we describe the integration of several instruments, including the Labcyte Echo 525 25 acoustic liquid handler and the iSeq and NovaSeq Illumina sequencing platforms, to miniaturize 26 and automate mNGS library preparation, significantly reducing the cost and the time required to 27 prepare samples. Through the use of External RNA Controls Consortium (ERCC) spike-in 28 RNAs, we demonstrated the fidelity of the miniaturized preparation to be equivalent to full 29 volume reactions. Furthermore, detection of viral and microbial species from cell culture and 30 patient samples was also maintained in the miniaturized libraries. For 384-well mNGS library 31 preparations, we achieved a savings of over 80% in materials and reagents alone, and reduced 32 preparation time by 90% compared to manual approaches, without compromising quality or 33 representation within the library. 34 35 3 36 Introduction 37 Metagenomic next-generation sequencing (mNGS) is becoming an increasingly useful tool in the 38 field of biology and clinical medicine. Its applications are almost limitless -any nucleic acid 39 can be turned into a library, amplified, and sequenced, making mNGS an appealing technology 40 for labs and hospitals alike. As sequencers such as the Illumina NovaSeq increase throughput, 41 hundreds to thousands of libraries can be sequenced in a single run. Although the per-base cost 42 of sequencing has become less expensive over the last several decades, the cost and time 43 associated with sample preparation remain disproportionately high [1,2]. 44 45 Manual library preparation is tedious and is often the bottleneck for many sequencing projects. 46 Numerous library preparation protocols have been adapted for automation through the use of 47 various positive displacement tip-based liquid handler instruments, including the Beckman 48 Coulter Biomek, Hamilton Star, Agilent Technologies Bravo, TTP LabTech Mosquito, and 49 others [3-5]. Though these provide more hands-off time during the library preparation process, 50 the overall cost can often exceed that of hand-prepared libraries due to the increased dead 51 volume of reagents and the large number of expensive, sometimes proprietary tips required for 52 liquid handlers. Furthermore, sub-microliter miniaturization is a challenge for the majority of 53 positive displacement based liquid handlers. 54 55 Recently, ac...

show abstract

“…Calibration of biological assays will also aid in comparing results within a single laboratory over time and across different laboratories. Recent studies, for example, for fluorescence [62,63], absorbance [64], and RNAseq [65] measurements, demonstrate the possibility of realizing scalable and cost-effective comparability in biological measurements. Best practices from biomanufacturing may be of use in informing all of these decisions.…”

Section: Testing the Function Of Engineered Genomesmentioning

confidence: 99%

Organizing genome engineering for the gigabase scale

et al. 2020

View full text Add to dashboard Cite

Engineering the entire genome of an organism enables large-scale changes in organization, function, and external interactions, with significant implications for industry, medicine, and the environment. Improvements to DNA synthesis and organism engineering are already enabling substantial changes to organisms with megabase genomes, such as Escherichia coli and Saccharomyces cerevisiae. Simultaneously, recent advances in genome-scale modeling are increasingly informing the design of metabolic networks. However, major challenges remain for integrating these and other relevant technologies into workflows that can scale to the engineering of gigabase genomes.In particular, we find that a major under-recognized challenge is coordinating the flow of models, designs, constructs, and measurements across the large teams and complex technological systems that will likely be required for gigabase genome engineering. We recommend that the community address these challenges by 1) adopting and extending existing standards and technologies for representing and exchanging information at the gigabase genomic scale, 2) developing new technologies to address major open questions around data curation and quality control, 3) conducting fundamental research on the integration of modeling and design at the genomic scale, and 4) developing new legal and contractual infrastructure to better enable collaboration across multiple institutions.

show abstract

External RNA Controls Consortium Beta Version Update

Cited by 32 publications

References 7 publications

Nanopore direct RNA sequencing maps an Arabidopsis N6 methyladenosine epitranscriptome

Nanopore direct RNA sequencing maps an Arabidopsis N6 methyladenosine epitranscriptome

Miniaturization and optimization of 384-well compatible metagenomic sequencing library preparation

Organizing genome engineering for the gigabase scale

Contact Info

Product

Resources

About