ONT long-read genome assembly with dual assembler approach (Pieridae)

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Method synopsis

Oxford Nanopore long-read sequencing followed by assembly using both FLYE and NECAT assemblers, with iterative polishing using Racon and Medaka, then merging assemblies using Quickmerge and HaploMerger2.

Synthesized from method descriptions across 2 papers using this protocol.

Procedure as described in the canonical source

Steps below were extracted from the paper that introduces this protocol — The genome of the Margined White butterfly (Pieris macdunnoughii): sex chromosome insights and the power of polishing with PoolSeq data (2021), Genome Biology and Evolution. Implementations in other papers (listed below) may differ.

1. Oxford Nanopore sequencing
   Isolated DNA was sequenced on a MinION platform, with fast base-calling using GUPPY (v.3.2.10) in the miniKNOW (v.3.6.5) software. A total of 3.82 million reads (28.17Gb) were generated from one flow cell providing ~90X coverage. The N50 length of the fast base-called subreads was 17.24 kb.
   Quantities: 3.82 million reads, 28.17Gb total, ~90X coverage, N50 of 17.24 kbDuration: Single flow cell runConditions: MinION platform operation
   Equipment: MinION platform, GUPPY (v.3.2.10), miniKNOW (v.3.6.5)
2. Assembly generation
   Generated three assemblies from raw reads: fast base-calling assembled with Flye (v.2.7), high-accuracy base-calling assembled with Flye and high-accuracy base-calling assembled with NECAT (v.0.0.1). For both Flye and NECAT, genome size was set as 300mb. Flye was run with the -meta option and two polishing iterations. Four rounds of Racon (v.1.4.13) on each assembly, followed by Medaka (v.1.0.3) to polish assemblies with nanopore data using default settings.
   Quantities: Three initial assemblies, four rounds of Racon polishing eachDuration: Multiple computational runsConditions: Computational analysis
   Equipment: Flye (v.2.7), NECAT (v.0.0.1), Racon (v.1.4.13), Medaka (v.1.0.3)
3. Assembly consolidation
   Consolidated haplotype redundancies using PURGEhaplotigs (v.1.0.3, default settings). Used Quickmerge (v.0.3) to merge the polished and purged Flye (self_assembly, -l 197436) and NECAT (hybrid_assembly) assemblies. Used HaploMerger2 (v.20180603) to collapse duplicated haploid content in the Quickmerge assembly.

Typical Equipment

• MinION platform
• GUPPY (v.3.2.10)
• Flye (v.2.7)
• NECAT (v.0.0.1)
• Racon (v.1.4.13)
• Medaka (v.1.0.3)
• Quickmerge (v.0.3)
• HaploMerger2 (v.20180603)

Output Measurements

• draft genome assembly
• contig statistics
• N50 values

Papers Using This Protocol (2)

Species Studied With This Protocol

Places Where Used