Nanopore Sequencing

ONT Ligation Other Applications

Oxford Nanopore Technologies (ONT) produces a long read platform designed to sequence genetic material of any length and is capable of generating the longest reads of any Next Generation Sequencing (NGS) platform. ONT’s unique nanopore sequencing technology measures the flow of ionic current across thousands of nano-sized protein pores. Bases are called according to voltage changes as nucleic acid molecules pass through each pore. This captured kinetics data stores information used not only for basecalling but also to predict base modifications with current (and future) models. In addition to DNA, the ONT platform is able to process and sequence both RNA and proteins.

One of the largest advantages of the Nanopore platform is that read length output is determined by the length of the library molecule, with the potential to sequence the full length of a chromosome in a single piece. Long read length is key in providing scaffolding information for de novo assemblies and can be sequenced straight through repeat regions that traditionally stymy short-read-only approaches. As a standalone, Nanopore sequencing can be used to resolve complex structural rearrangements common with insertions and mobile elements or when verifying constructs. While this technology has a slightly lower per base accuracy than our other platforms, it is easily overcome when paired with supplemental Illumina short read sequencing for a hybrid approach or through deep coverage.

The Nanopore sequencing community is also very active and continues to develop a plethora of third-party bioinformatic tools to get the most out of the unique data provided for the wide breadth of projects it can support.

As an accredited ONT sequencing provider, we are proud to offer ligation-based DNA sequencing that makes the most of native molecule libraries and are excited to support the full range of ONT library preparations for custom applications.

  • ONT Ligation Icon

    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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  • Other Applications

    ONT offers a wide array of library preparations that we are happy to support, including direct RNA sequencing which is unique to the ONT platform.

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We are also happy to accommodate sequencing of prepared ONT libraries. Please contact us for custom handling and refer to our ONT Ligation page for flowcell pricing.

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

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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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