Direct RNA

ONT Ligation Direct RNA Ultra-Long DNA

Oxford Nanopore Technologies’ Direct RNA Sequencing Kit (SQK-RNA004) offers direct sequencing of native polyadenylated (poly-A) mRNA molecules, without the need for reverse transcription or PCR. This enables researchers to preserve the natural length and chemical modifications of RNA, while capturing strand-specific, full-length transcript data in real time. The long-read capability inherent in nanopore sequencing allows for comprehensive isoform resolution, identification of alternative splicing events, and the detection of structural variation in RNA, all of which often go undetected with short-read technologies. Due to the specialized nature of this technology, sample multiplexing is not supported and requires each order be sequenced on its own dedicated flowcell.

Moreover, by sequencing RNA in its native state, the Direct RNA Sequencing Kit uniquely enables the detection of RNA base modifications, such as N6-methyladenosine (m6A), directly from raw signal data—providing a powerful tool for studying the epitranscriptome without the need for indirect enrichment or modification-specific assays.

The versatility of this method makes it well-suited for a wide range of research applications, including novel transcript discovery, viral RNA characterization, single-gene expression profiling, and comparative transcriptomics across model and non-model organisms. Importantly, this approach does not rely on existing genome annotations, enabling reference-free transcriptome analysis, which is especially valuable for emerging pathogens, poorly characterized species, or complex environmental samples.

In order to obtain high-quality and biologically meaningful data from an RNA-seq experiment, careful experimental design is critical. In particular, please consider details such as sample preparation and storage, the level of biological replication, sequencing read depth, and batch effects.

  • Ensure the sample remains as cold as possible (avoiding freeze/thaw cycles) from the moment of collection. This will maximize integrity and preserve expression profiles.
  • Take precautions to shield samples from RNases, which are ever-present.
  • Replication is arguably more important than read depth or read length in many power analyses. When determining the proper number of replicates, some factors to consider are effect size, acceptable false-positive and false-negative rates, and maximum sample size.
  • Ship RNA on dry ice with single-day overnight air delivery. Please consider SeqCenter operating hours when shipping as our receiving is closed on the weekends.

Sample Requirements:

  • Total volume of 20 µL or more
  • Eluted into nuclease-free water
  • Sample concentration greater than:
    • Pre-depleted, polyadenylated RNA: at least 300ng (15ng/µL)
    • Undepleted, polyadenylated RNA: at least 1000ng (50ng/µL)
    • (Optional) RIN > 8 for best results

What You Will Receive:

  • One fastq file for each sample
  • One BAM file with 5mC calls for each sample
    • Alternative basecalling models available upon request (Dorado)
  • Pod5 files with kinetics data
  • Sequencing stats summary
  • Materials and methods summary
  • Data shared via Globus within 2 weeks.

Packages & Pricing

Sequencing Package
(Minimum Read Count Per Sample)		 Price*
Dedicated GridION Flowcell
Includes 1 Library Prep. sequencing on a full GridION flowcell		 $1,200.00
*Orders >48 Samples receive 5% discount

Due to the sensitive nature of RNA and RNA-seq projects, we recommend an approach tailored to a project’s organism and experimental setup that is carried out in a uniform manner soon after harvesting.

For customer convenience, we do offer RNA extraction services using the Zymo Quick-RNA Miniprep Kit, which is broadly applicable but may not be optimal for all sample types or projects. This kit enables us to quickly isolate DNA-free RNA from cell pellets as well as homogenized tissue samples. We recommend sending cell pellets that contain at least 107-108 cells, in which the pellet is sizable and clearly visible in the tube. For tissue samples, we ask that you homogenize them as much as possible to enable successful extractions.

This extraction kit is compatible with ZymoResearch’s DNA/RNA Shield, and it should be noted on orders when samples have been treated with this product. More info on the Quick-RNA Miniprep Kit can be found here.

Detection of RNA modifications and polyA tail length in ONT data

Direct RNA sequencing of native molecules has the same added benefits of native DNA sequencing, including being able to capture kinetics data that can be plumbed for modified base motifs and polyadenylated tails.

The nanopore community continues to develop a host of third-party tools to analyze the kinetics data for much more than just basecalls for many different applications, including a know-how page for polyA-tail length estimation. (Tool requires free ONT account.) Though ONT does not currently offer in-house tools directly for RNA modifications, their current tools are being tested for RNA applications. The company has hosted research groups presenting their use and analysis for nanopore data, including m6A modification detection in human neuroblastoma cell lines.

Additional resources:

ONT Ligation

Ligation-based library preparation and native DNA molecule sequencing take advantage of the potentially unlimited read length of the ONT platform to deliver Q20+ quality data in lengths limited only by the input molecules. We offer both standalone ONT sequencing as well as hybrid packages, which include high-accuracy Illumina sequencing and de novo assembly.

Explore

Ultra-Long DNA

This transposase-based library preparation chemistry with rapid adapter attachment allows reliable capture of long DNA fragments. Bring high-resolution, long-range insights into reach and go beyond the standard ONT Ligation service.

Explore

Contact:
91 43rd Street, Ste. 250
Pittsburgh, PA 15201
(878) 227-4915

Services:

About:

Resources:

Full Flowcell:

We offer full flowcell sequencing services for libraries that have been prepared outside of our lab. For each library, we will need to know the loading concentration, phiX spike-in, and run structure. Data is delivered as a complete run folder and can be demultiplexed upon request if a sample sheet is provided.

SARS-CoV-2 Sequencing:

Our SARS-CoV-2 Sequencing services include an assembly on the SARS-CoV-2 reference genome as a scaffold. These packages provide roughly 200Mbp of paired end reads (2×151 bp) as fastq files and the assembly in fasta format.

We require a minimum sample volume of 15 µL of extracted material (or 250µL for VTM samples) and recommend that only samples with Ct values below 35 are sent (because submissions are not measured for viral load before processing). We offer services for both purified RNA and samples in VTM media that require viral RNA extraction. Please see our SARS-CoV-2 Sequencing page for more detailed information.

Microbiome Sequencing:

Please see our Microbiome Sequencing page for package details, including distributables and sample requirements.

Nanopore Sequencing:

Our Oxford Nanopore sequencing packages provide reads equal to the length of input DNA, delivered as a fastq file.

For each sample, we require a minimum volume of 60 µL, and at least 40 ng/µL dsDNA quantified by Qubit or other dye-based method (100 ng/µL by Nanodrop). Variant calling add-on analysis requires an annotated reference file. DNA extraction services are available for only specific categories of microbes.

Please see our DNA Sequencing: Long Read Sequencing page for more detailed information about the package, variant calling analysis, and extraction requirements.

PacBio:

Our PacBio services deliver HiFi reads with an average read length of 8-12kb for standard offerings and up to 25kb for full flowcell runs. All package names are listed by the minimum number of >Q20 reads that will be delivered. Both assembly and annotation are included with all packages.

For each sample, a minimum volume of 60 µL and at least 35 ng/µL dsDNA (quantified by Qubit or other dye-based methods) are required. SeqCenter strongly recommends utilizing our HMW DNA Extraction service for best results; however, we will also accept samples that are shipped on dry ice to avoid DNA integrity issues caused by the shipping process.

Please see our PacBio Sequencing page for more detailed information about our services.

RNA Sequencing:

We offer both rRNA depletion RNA squencing and polyA-tail enrichment mRNA sequencing. Both provide stranded, paired end reads (2x 151bp) as fastq files.

For each sample, we require a minimum volume of 20µL and at least 50ng/µL RNA. We also recommend, but do not require, that samples be DNAse treated and have a RIN score greater than 6. We also offer Basic and Intermediate RNAseq analyses, which require an annotated reference sequence.

Please see our RNA Sequencing page for more detailed information about both our RNA sequencing and analysis packages.

Illumina Sequencing:

The Illumina sequencing packages produce paired end reads (2×151 bp) delivered as fastq files.

For each sample, we require a minimum volume of 30 µL and at least 10 ng/µL dsDNA quantified by Qubit or other dye-based method (20 ng/µL by Nanodrop). Variant calling add-on analysis requires an annotated reference file. DNA extraction services are available for only specific categories of microbes.

Please see our DNA Sequencing: Whole Genome Sequencing page for more detailed information about the package, variant calling analysis, and extraction requirements.

For additional information about variant calling and short read assemblies, please see our Bioinformatics page.

Go to Top