Long Read Sequencing

Genomic, transcriptomic and epigenetic characterization with high precision

Why choose long read sequencing?

Next generation sequencing (NGS) technologies typically make use of generating 75-300bp long reads, which are then either de-novo assembled in a genome or mapped to a reference genome. Complex regulatory elements or highly repetitive sequences can make this mapping harder than for uniquely variable ones. With long read sequencing more than 100.000 bp long reads are assembled, making it easier to tackle:

Highly contiguous de novo assemblies
Sequencing through extended repetitive regions
Discovery of novel genes and novel isoforms of annotated genes

Long read sequencing is also frequently used in combination with short read sequencing.

long read sequencing advantages Continuous Long Read Sequencing

Mirjam Rademaker Continuous Long Read Sequencing

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PacBio (Pacific Biosciences) and Nanopore (Oxford Nanopore Technologies: ONT)

GenomeScan offers long read sequencing based on two distinct technologies, each with its own set of advantages.

Long read lengths up to ~25kb long for PacBio HiFi
Throughput per Sequel IIe SMRT cell: ~ 26 Gb, Revio SMRT cell: ~80 Gb
High read accuracy Q30 >99,9%

Long read lengths up to > 4Mb
1 PromethION Flowcell: ~100-200 Gb
Kit 14 Chemistry > Q20 (99%) for Simplex reads, ~Q30 (99.9%) for Duplex reads

Sequencing with PacBio

By utilizing SMRT technology, we are able to easily generate highly accurate (median consensus accuracy of >99.9%) long reads up to 30 kb (‘CCS or ‘HiFi’ reads). With reads tens of kilobases in length you can readily assemble complete previously uncharacterized genomes, sequence full-length transcripts as well as genes or regions of interest ranging in size from hundreds base pairs to 30 kb.

For this service we have available Sequel IIe and Revio System.

	Revio System	Sequel IIe System	Increase
Higher density	25M ZMW	8M ZMW	3x
Independent stages	4	1	4x
Shorter run times	24hs	30hs	1.25x
CCS data	~ 80Gb	~20GB	4x

Additionally, calling and detection of variants benefit from long-read sequencing thanks to low sequencing-context bias, and accurate mapping of reads. Single Molecule, Real-Time (SMRT) Sequencing is a core technology and a powerful tool for analyzing variable number tandem repeats, or for repeat occurrence with disease significance, such as in Huntington’s disease.

Easier assembly of complete genomes
Sequencing full-length RNA transcripts
Direct detection of epigenetic modifications
Haplotype phasing information
Base-level resolution with >99.9% single-molecule read accuracy
Unbiased coverage of GC rich regions such as promoters
No PCR amplification, prevention of errors

How it works

Sequencing with Oxford Nanopore

Nanopore sequencing involves reading the code of single DNA/RNA strand as they are threaded through nanopores embedded within a membrane. As the DNA/RNA moves through the pore, the current is disrupted to produce a characteristic ‘squiggle’. The squiggle is then decoded using basecalling algorithms to determine the DNA or RNA sequence in real time.

Advantages:

Direct seq of DNA and RNA without using PCR: No bias, richer info including epigenetics.
Read length-agnostic: It’s able to seq different length of genome.
Real-time analysis: Rapid results, adaptive sampling, base calling at the same time its sequencing.
Chemistry on bespoke electronics: Scalable, from small to large formats.

ONT explanation 2 Continuous Long Read Sequencing

How it works

Which technology to choose

Nanopore’s defining feature is its real-time analysis, while PacBio promises high fidelity reads. If you are unsure about which technology suits your project the best, our specialists would be happy to help you.

In some cases, a hybrid sequencing strategy that combines different approaches (i.e. long read and short read sequencing) may be your perfect solution to answer complex questions.

Data analysis

GenomeScan’s bioinformatics experts apply optimized pipelines for preprocessing, assemblies, variant calling and disease annotations to analyze your dataset. With our customizable workflows, we help you receive reliable and publication-ready results.

Bioinformatic pipelines can also be tailored according to your research or clinical needs allowing more in-depth mining of your dataset.

Long Read Sequencing Resources

Policies

FAQ

Sample
submission form

Publications

Service workflow

1. Initial meeting

When you consider using one of our services, our scientific team will help choosing the optimal project design. Each service can be customized by adding options. You will receive an overview of project budget and deliverables, so you can make an informed choice. Once you return your Purchase Order form, your project manager will contact you to discuss the next steps.

2. Sample delivery

Sample specifications for long read sequencing are:

Purified DNA

Quality: High molecular weight DNA (HMW) and OD260/280 ~1.8-2.0 ; OD260/230 ~2.0-2.2
Validated input/ Minimum volume: application dependent (the scientific team will inform you accordingly based on the type of project)

3. Sample entry QC

After sample arrival, Entry-QC is performed to determinate the sample quality and quantity.

A report with QC results will be provided and results and progression of the project can be discussed.

4. Library prep/QC and sequencing

The library resulting from sample preparation goes into a library QC step to ensure sufficient material per sample and the right library size.

5. Data QC

The raw data that is generated by the sequencer must be divided to obtain one data file per sample. This is called demultiplexing.

Your project manager checks the data quality by analyzing the quality metrics of the run. Final inspection of the data takes place and the data is either transferred on a hard disk or it is uploaded to the GS customer portal.

6. Reporting

You receive your Project Report together with your data. It contains an overview of the experimental methods used, sample specifications, and a summary of all technical information for publication. Deliverables:

Raw data files via secure e-transfer or shipment on storage medium
Quality score Q30 of ≥80% for PE150 reads
Optional data analysis with comprehensive report

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long read sequencing product specification Continuous Long Read Sequencing

Service specifications

We have summarized key information about our long-read sequencing service into a service specification sheet.

Download sheet

Other Services

Whole Genome Sequencing

Whole Exome Sequencing

Gene expression profiling

Epigenetics

Single cell sequencing

Targeted proteomics panel

Let's get the conversation started for your next NGS project