Nanopore Sequencing and Genome Assembly

Nanopore sequencing of 2 μg of gDNA was prepared using an NEB Next FFPE DNA Repair Mix kit (New England Biolabs) and subsequently processed using the ONT Template prep kit (Oxford Nanopore Technologies). The large segment library was premixed with loading beads and then pipetted onto a previously used and washed R9 flow cell. The library was sequenced on the ONT PromethION platform with the Corresponding R9 cell and ONT sequencing reagents kit (EXP-FLP001.PRO.6; Oxford Nanopore Technologies). The genome was assembled based on three ways: initial wtdbg2 (Ruan and Li, 2020 (link)) assembly and then SMARTdenovo assembly (Liu et al., 2021 (link)), followed by error correction using racon (Vaser et al., 2017 (link)) software and adjustment by Pilon (Walker et al., 2014 (link)) software. The assembly results were evaluated by the ratio of sequencing reads, Core Eukaryotic Genes Mapping Approach (CEGMA; Parra et al., 2007 (link)), and Benchmarking Universal Single-Copy Orthologs (BUSCO; Simão et al., 2015 (link)).

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Wang F., Zhang B., Wen D., Liu R., Yao X., Chen Z., Mu R., Pei H., Liu M., Song B, & Lu L. (2022). Chromosome-scale genome assembly of Camellia sinensis combined with multi-omics provides insights into its responses to infestation with green leafhoppers. Frontiers in Plant Science, 13, 1004387.

Publication 2022

NEB Next FFPE DNA Repair Mix kit ONT Template prep kit

Cell Dna repair Eukaryotic Genes Genome Library Walker

Corresponding Organization :

Other organizations : Qiannan Normal College For Nationalities, Ministry of Natural Resources, Guizhou Academy of Agricultural Sciences, Guizhou University, Biomarker Technologies (China), Peking University

Top 5 similar protocols

Variable analysis

independent variables

Nanopore sequencing of 2 μg of gDNA
NEB Next FFPE DNA Repair Mix kit (New England Biolabs)
ONT Template prep kit (Oxford Nanopore Technologies)
R9 flow cell
ONT PromethION platform
Corresponding R9 cell and ONT sequencing reagents kit (EXP-FLP001.PRO.6; Oxford Nanopore Technologies)
Wtdbg2 assembly
SMARTdenovo assembly
Racon software
Pilon software

dependent variables

Genome assembly quality
Ratio of sequencing reads
CEGMA (Core Eukaryotic Genes Mapping Approach) evaluation
BUSCO (Benchmarking Universal Single-Copy Orthologs) evaluation

control variables

Previously used and washed R9 flow cell

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