Ultra-Long DNA

ONT Ligation Direct RNA Ultra-Long DNA

Oxford Nanopore Technologies’ Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114) is specifically designed to generate ultra-long sequencing reads from ultra-high molecular weight (uHMW) DNA. As part of the sequencing workflow, each sample undergoes a quality check upon arrival, including size selection and fragment analysis, to ensure that only the longest DNA fragments proceed into library preparation. Utilizing a transposase-based library preparation method and rapid adapter attachment technology, this kit enables efficient, reliable processing of ultra-long DNA while minimizing fragmentation and preserving native epigenetic modifications. Due to the specialized nature of this technology, sample multiplexing is not supported, requiring each order be sequenced on its own dedicated flowcell.

The Ultra-Long Kit V14 is exceptionally well-suited for a wide range of advanced applications, including: de novo genome assembly, where long reads help span repetitive regions and close gaps; telomere-to-telomere (T2T) sequencing, enabling the resolution of entire chromosomes from end to end; structural variant discovery, where large insertions, deletions, and rearrangements can be accurately detected; and epigenetics and methylation studies, which benefit from the preservation of native base modifications through direct, amplification-free sequencing.

With a streamlined protocol, the Ultra-Long Kit V14 brings high-resolution and long-range insights within reach for every lab aiming to go beyond conventional sequencing limits.

  • The ONT Ultra-Long sequencing pipeline has more stringent submission requirements relative to our other Nanopore services.
  • The extraction and clean-up methods used often have the greatest influence on final yield and sample quality in terms of purity and fragment length.
  • Please consider that multiple extraction attempts may be required to meet sample submission requirements.

Sample Requirements:

  • Total volume of 750 µL or more
  • Eluted into water or dilute Tris/TE buffer
  • Sample concentration greater than:
    • 30-40 ng/µL measured by Qubit

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*
Ultra-Long Sequencing on a GridION Flowcell
Includes standard Femto Pulse Fragment Analysis and XL SRE Kit processing		 $1,350.00
Ultra-Long Sequencing on a PromethION Flowcell
Includes standard Femto Pulse Fragment Analysis and XL SRE Kit processing		 $2,250.00

To help our customers meet the strict sample requirements, SeqCenter offers convenient HMW DNA extractions using PacBio’s Nanobind PanDNA kit for a fee. Please note that if additional sample homogenization is needed (e.g. using a TissueRuptor,) are additional extractions are needed to meet minimum input requirements, additional fees will applies.

We are able to accommodate organisms up to BSL2+ and do not accept BSL3 or higher classified organisms for extraction services. We cannot culture samples and encourage you to ensure samples are fresh, pure and that there is adequate growth (if applicable) on your plates before shipping. Minimum sample input requirements for extractions can be found in the table below. While we have had success using this kit with many sample types, this kit is not broadly applicable. Please reach out to info@seqcenter.com to confirm kit compatibility with your sample type.

Sample Type
Starting Material		 Minimum Sample Input
Blood
Human whole blood		 200 µL
Blood
Nucleated red blood cells (nRBCs)		 20 µL
Blood
Human whole blood with RBC lysis		 400 µL
Animal Tissue
Diverse types		 100 mg
Insect tissue
Whole body or segments		 30 mg
Plant tissue
*Isolated plant nuclei		 5 mg
Mammalian cultured cells
Suspension and adherent cell culture		 5×106 diploid cells
Cultured bacteria
Gram-positive and gram-negative		 5×106 bacterial cells

*Plant nuclei isolation available upon request.

We offer a selection of basic bioinformatic services to get you started with analyzing your data. These services are meant to be the first step into subsequent investigation and extensive validation studies and include de novo long read assembly and annotation services. For more information on these services or custom services like reference-based scaffolding or integrating additional sequencing data into a de novo assembly, please see our bioinformatics service pages.

Detecting Nucleotide Modifications in ONT Data

ONT sequencing generates kinetics data for each native DNA molecule as it passes through a nanopore by measuring the electrical current flowing across it. By interpreting the changes in current, this data is used not only to call bases but also to later predict base modifications. At this time, 5mC metadata is generated during basecalling, stored in the returned bam files, and can be later used to predict 5mC methylation sites with tools.

While there are only a few tools offered through ONT directly, namely Remora and Dorado, there is a significant community of researchers working on developing new tools for predicting additional modifications from this data. This is a curated list of some of the available open-source tools the community has created, including for modification analysis.

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.

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Direct RNA

Directly sequencing native RNA without reverse transcription or PCR preserves the natural structure, length, and chemical modifications of RNA. This full-length transcript data allows for comprehensive isoform resolution, identification of alternative splicing events, epitranscriptome characterization, and the detection of structural variation in RNA.

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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.

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