T. H. (Henry) Hung [email protected], University of Oxford

Why Nanopore Sequencing?

• The four leading sequencing technologies are Nanopore, PacBio CLR, PacBio CSS (also known as HiFi sequencing), and Illumina
• Nanopore (chemistry 14) is comparable to PacBio CSS and Illumina in terms of sequencing accuracy
  • Nanopore chemistry 14 has a Q-score ≥20 (99%), while PacBio CSS and Illumina ≥30 (99.9%) and PacBio CLR ≥10 (90%)
  • Consensus accuracy after correction can be near-perfect
• Nanopore outperforms PacBio CSS and is comparable to Illumina in terms of sequencing throughput
  • Nanopore outputs ≥5-fold more than PacBio CSS
• Nanopore outperforms PacBio CSS mode and Illumina in terms of read length
  • Nanopore N50 ≥ 50 Kbp (max 5 Mbp), while PacBio CSS 10**–**25 Kbp and Illumina < 1 Kbp

https://www.youtube.com/watch?v=E9-Rm5AoZGw

Nanopore Ligation-based Sequencing

• The highest throughput, read length, and accuracy

• Minimum sequencing coverage:

  • 30x for each de novo genome assembly or re-sequencing
  • 10x for hybrid genome assembly (e.g. 50x Illumina sequencing is matched)

• Cost:

  • £90 / library preparation
  • Excluding delivery cost for purchase of materials and VAT

• Input requirement

  • 3 μg high molecular weight genomic DNA (HMW-gDNA), size-selection preferred

• Optimisations

  • HMW-gDNA must be extracted using methods that have minimal shearing

  HMW DNA Extraction from Plant Tissues for (Ultra-)Long-Read Sequencing

  • Size selected with Short Read Elimination Kits to enhance read length
    • SRE-XS ≥10 Kbp
    • SRE ≥25 Kbp
    • SRE-XL 40 Kbp (recommended)

Pore-C Proximity Ligation

• Anchoring long-read contigs to produce chromosome-level assemblies

• Minimum sequencing coverage:

  • 10x for each genome

• Cost and preparation:

  • Please follow this protocol:

  Pore-C Preparation for Plant Foliage Tissues

  • Each Pore-C proximity ligation product needs 1 Rx of Ligation-based sequencing (see above): £ 90 / library preparation

• Input requirement

  • 1 μg Pore-C product

Sequencing & Multiplexing

• Multiplexing can be achieved to sequence multiple samples on the same flow cell using Native Barcoding Kits