Other Applications

ONT Ligation Other Applications

The ONT sequencing platform offers countless uses for the nanopore system, with library preparation and downstream analyses to match that go beyond our standard ligation DNA sequencing. As a certified ONT service provider, we have the ability to order any library preparation that is offered for this system if a project calls for it, and the nanopore platform has a diverse range of applications that take advantage of its unique features, relative to other platforms. While the possibilities are numerous, an example library preparation and analysis are listed below. If you have a custom project that is well-suited to the nanopore platform, please contact us at info@seqcenter.com. We would love to discuss your project’s goals and how SeqCenter can support your research.

Direct RNA sequencing

The nature of the nanopore platform affords it the unique ability to perform direct RNA sequencing, which is not available from any other major sequencing technology. ONT offers a Direct RNA Sequencing Kit and protocol suitable for total RNA or enriched samples. It is great for reducing any bias introduced during cDNA synthesis and detection downstream of modified bases.

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: capture of 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.

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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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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