ONT Ligation

ONT Ligation Other Applications

Oxford Nanopore Technology (ONT) Ligation Sequencing library preparation with Native Barcode kit provides a median raw read accuracy of Q20+ (99%) with a protocol that is free from PCR, fragmentation, and size selection. There are numerous advantages to this approach, one of which is the elimination of PCR error and enzyme bias. Additionally, nanopore sequencing provides direct sequencing of native DNA molecules which can produce extremely long reads, as well as kinetic data for each molecule to infer base modifications of the nucleic acids.

Sequencing of native molecules does impose higher input requirements compared to PCR-based methods because the ligation-based preparation is less efficient. This effect is compounded further by the lack of amplification. Additionally, the nanopore platform is sensitive to certain compounds that can be carried over during extraction, such as polysaccharides (common for some fungi, plants, and insects) and excessive amounts of unprepared nucleic acids (e.g., excess RNA in a DNA library).

SeqCenter aims to provide nanopore sequencing in packages and prices that are applicable and affordable for projects of all sizes, including small genomes. By multiplexing all of our single sample packages, we are able to subdivide the output and cost of the nanopore flowcells for both our hybrid sequencing and ONT-only services.

We recommend taking steps to maximize DNA fragment lengths when extracting and handling to optimize long read sequencing platforms.

Often, the biggest determinant of fragment length is the DNA extraction process. Gentle extraction methods like enzyme-based kits often produce the longest read lengths. However, some tough-to-lyse cells may require physical or thermal lysis methods for adequate DNA extraction that can come at the cost of shorter DNA fragments. Some sample types may need their extraction methods to be fine-tuned to strike the right balance.

SeqCenter will attempt to sequence all samples submitted and does not impose a minimum length requirement. We encourage our customers to assess fragment length before sending samples.

Handling and storage will also affect DNA integrity, especially during shipping. We recommend avoiding freeze-thaw cycles and other extreme changes to minimize degradation. If DNA samples are already frozen, we recommend shipping on dry ice (with 5-10 lbs per day of expected transit). If possible, liquid samples should be shipped on ice packs to buffer against temperature changes, especially during summer months.

Hybrid Sequencing and Assembly

ONT long read sequencing is commonly used to provide the backbone for de novo assemblies, and for good reason. The nanopore platform provides an economical way to generate much needed structural information and resolution of repetitive motifs. When combined with high-accuracy Illumina short reads, nanopore sequencing is a cost-effective tool in closing assemblies of less complex genomes, like bacteria or fungi.

SeqCenter offers this powerful hybrid approach as “Nanopore Combos” that include ONT long read sequencing, Illumina short read sequencing, and programmatic de novo assembly and automation services. These packages aim to produce reference-quality assemblies and are well-suited for clonal bacterial or fungal samples.

Sample Requirements:

  • Two aliquots of sample:
    • Illumina aliquot:
      • Total volume of 30 µL or more
      • Sample concentration greater than:
        • 10 ng/µL measured by Qubit
        • 20 ng/µL measured by Nanodrop
    • Nanopore aliquot:
      • Total volume of 40 µL or more
      • Sample concentration greater than:
        • 40 ng/µL measured by Qubit
        • 100 ng/µL measured by Nanodrop
  • Eluted into water or dilute Tris/TE buffer

What You Will Receive:

  • Raw Illumina data, including fastq files
  • Raw Nanopore data, including fastq, bam, and fast5 files
  • de novo Assembly and annotation in GenBank (.gbk, .gff3) and fasta (.fasta) formats
  • Sequencing and assembly stats summary
  • Materials and methods summary
  • Data shared via Box folder within 2 weeks

Packages & Pricing

Sequencing Package
(Minimum Read Count Per Sample)		 Price*
Small Nanopore Combo (Ideal for Genomes <10Mb)
Minimum 300 Mbp Long Reads, 400Mbp Illumina Reads, Genome Assembly and Annotation		 $350.00
Medium Nanopore Combo (Ideal for Genomes <25Mb)
Minimum 600 Mbp Long Reads, 1Gbp Illumina Reads, Genome Assembly and Annotation		 $450.00
Large Nanopore Combo (Ideal for Genomes <50Mb)
Minimum 1.2Gbp Long Reads, 2Gbp Illumina Reads, Genome Assembly and Annotation		 $600.00
*Orders >48 Samples receive 5% discount

For customer convenience, SeqCenter offers DNA extraction services using ZymoBIOMICS DNA extraction kits. While this protocol can extract DNA from a variety of sample types, it uses a bead bashing tube for consistent mechanical lysis and may not be the ideal extraction method for ONT sequencing as read length is often determined by the input.

We are able to accommodate all organisms up to BSL2+ and do not accept BSL3 or higher classified organisms for extraction services.

Samples requiring DNA extraction can be submitted as aspirated cell pellets shipped frozen on dry ice or as growth on sealed, nutrient agar plates shipped at ambient temperature. A minimum of 109 cells is recommended for all microbial DNA isolation submissions. We cannot culture samples; we encourage you to ensure samples are pure and that there is adequate growth before shipping.

Please note that while this kit is broadly applicable to many sample types, we cannot guarantee its efficacy for a given organism. More information about the ZymoBIOMICS kit can be found here.

Submission Considerations

The nanopore platform has a high minimum input requirement. Due to the high input requirement, two separate aliquots of DNA are required for hybrid sequencing with the Nanopore Combo services, one aliquot for each pipeline. We strongly recommend a dsDNA-specific, dye-based quantification method (like Qubit or PicoGreen) when assessing concentrations. If there is not enough material to complete the ONT portion, the hybrid package will be converted to the corresponding Illumina package, and only Illumina data will be delivered.

Standalone ONT Ligation Sequencing

SeqCenter also offers standalone ONT Ligation library preparation and sequencing services. Nanopore sequencing by itself can provide a cost-effective option for verifying structural modifications like large insertions, though higher depth may be required to confirm consensus sequences at the per-base level. Though the Q20 offers 99% and higher accuracy, if you are evaluating SNPs, we strongly recommend either adding short read Illumina sequencing or considering PacBio HiFi sequencing, as both offer average quality scores of Q30 (99.9%+) or higher which are better suited for verifying single nucleotide changes.

These nanopore-only packages are also a great way to supplement previous sequencing or assemblies. The addition of long read data can help close down the number of contigs when assembling with previously generated Illumina data and can add 5mC metadata to an existing assembly for later methylation prediction.

Sample Requirements:

  • Total volume of 60 µL or more
  • Eluted into water or dilute Tris/TE buffer
  • Sample concentration greater than:
    • 40 ng/µL measured by Qubit
    • 100 ng/µL measured by Nanodrop

What You Will Receive:

  • One fastq file for each sample
  • One BAM file with 5mC calls for each sample
  • Fast5 files with kinetics data
  • Sequencing stats summary
  • Materials and methods summary
  • Data shared via Box folder within 2 weeks.

Packages & Pricing

Sequencing Package
(Minimum Read Count Per Sample)		 Price*
300Mbp
Minimum 300Mbp Nanopore Reads Only		 $250.00
600Mbp
Minimum 600Mbp Nanopore Reads Only		 $320.00
1.2Gbp
Minimum 1.2Gbp Nanopore Reads Only		 $420.00
Dedicated GridION Flowcell
Includes 1 Library Prep. Full GridION Flowcell, Typical Output 10-20Gbp		 $1,100.00
Dedicated PromethION Flowcell
Includes 1 Library Prep. Full PromthION Flowcell, Typical Output 60-120Gbp		 $2,000.00
*Orders >48 Samples receive 5% discount

For customer convenience, SeqCenter offers DNA extraction services using ZymoBIOMICS DNA extraction kits. While this protocol can extract DNA from a variety of sample types, it uses a bead bashing tube for consistent mechanical lysis and may not be the ideal extraction method for ONT sequencing as read length is often determined by the input.

We are able to accommodate all organisms up to BSL2+ and do not accept BSL3 or higher classified organisms for extraction services.

Samples requiring DNA extraction can be submitted as aspirated cell pellets shipped frozen on dry ice or as growth on sealed, nutrient agar plates shipped at ambient temperature. A minimum of 109 cells is recommended for all microbial DNA isolation submissions. We cannot culture samples; we encourage you to ensure samples are pure and that there is adequate growth before shipping.

Please note that while this kit is broadly applicable to many sample types, we cannot guarantee its efficacy for a given organism. More information about the ZymoBIOMICS kit can be found here.

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:

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.

Explore

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

Services:

About:

Resources:

Copyright 2024

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