Table 1 Comparison of plasmid sequence verification methods
From: Complete sequence verification of plasmid DNA using the Oxford Nanopore Technologies’ MinION device
Our Pipeline | Currin et al. [14] | Circuit-Seq [18] | OnRamp [19] | Sanger (Regulated Environment) | |
|---|---|---|---|---|---|
Plasmids per run | 1 | 576 | 96 | ≥ 30 | 1 |
Library Prep | Restriction Digest and Adapter Ligation | Tn5 barcoded | Tn5 barcoded | Tn5 or Restriction Digest and Adapter Ligation | Primer Design |
Flowcell | MinION R10.3 | MinION R9.4.1 | Flongle R9.4.1 | Flongle R9.4.1 | – |
Raw reads per plasmid | ~ 3,860,000 | ≥ 46 | ~ 1000 | ~ 934 | 2 × |
Basecalling | Bonito Duplex | Guppy | Guppy | Guppy | |
Reference-guided | No | Yes | No | Yes | No |
Consensus Generation | Assembly, Alignment and Pileup | Alignment and Pileup | Error correction, Assembly, Medaka Polishing | Medaka consensus | Phrap, Consed [29] |
Output | .fasta file + position-based nucleotide frequencies | .vcf file | .fasta file | .bam file | .fastq file |
Quality Metrics | Per-base signal:noise, coverage | Summary statistics for alignment | – | Quality score of consensus based on number of variants | Per-base PHRED scores |
Cost (~ 10 kb) | $1,500 | $3 | $1.50 | $15 | $15,000 |
Time | 7 days | 3 days | 1 day | 2 days | 25 days |