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Ultra-long Read Sequencing for Whole Genomic DNA Analysis
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Robust long-read native DNA sequencing using the ONT CsgG Nanopore system.

Jean-Michel Carter1, Shobbir Hussain1

  • 1Department of Biology and Biochemistry, University of Bath, Bath, BA2 7AY, UK.

Wellcome Open Research
|May 16, 2017
PubMed
Summary

Oxford Nanopore Technologies (ONT) sequencing with the new CsgG pore system and 250b/s chemistry shows promise for long DNA read lengths. This method achieved high throughput and accuracy, suggesting potential for practical applications in genomics.

Keywords:
CsgGHAP1Nanopore sequencingOxford NanoporeR9.4complex genomeslong read sequencing

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Area of Science:

  • Genomics
  • Molecular Biology
  • Bioinformatics

Background:

  • Long read lengths in DNA sequencing are crucial for various applications.
  • Oxford Nanopore Technologies (ONT) offers Nanopore sequencing, known for long reads but historically limited in throughput and accuracy.
  • Recent advancements include ONT's CsgG pore and 250b/s translocation chemistry, aiming to improve performance.

Purpose of the Study:

  • To evaluate the performance of ONT's CsgG pore sequencing system with 250b/s chemistry.
  • To assess the throughput, read length, and accuracy of this new Nanopore sequencing setup.
  • To explore its potential for practical long-read genomic applications.

Main Methods:

  • Utilized ONT's MinION device with CsgG pore and 250b/s chemistry.
  • Sequenced native genomic DNA from the HAP1 cancer cell line.
  • Analyzed data using computational tools specifically designed for nanopore/long-read sequencing.

Main Results:

  • Generated approximately 240,000 high-quality mapped reads (~2.3 billion bases) in a single run.
  • Achieved a mean read length of 9.6kb and an N50 of ~17kb.
  • Obtained 85% mean identity to reference, ~68X coverage of the mitochondrial genome, and 99.8% consensus identity for mtDNA reads.

Conclusions:

  • ONT CsgG-based sequencing demonstrates improved performance over previous iterations.
  • The system shows potential for practical long-read applications, especially with ongoing advancements in chemistry and instrumentation.
  • High accuracy for mitochondrial DNA sequencing was a notable finding.