Constructing a Reference Genome in a Single Lab: The Possibility to Use Oxford Nanopore Technology

Abstract

The whole genome sequencing (WGS) has become a crucial tool in understanding genome structure and genetic variation. The MinION sequencing of Oxford Nanopore Technologies (ONT) is an excellent approach for performing WGS and it has advantages in comparison with other Next-Generation Sequencing (NGS): It is relatively inexpensive, portable, has simple library preparation, can be monitored in real-time, and has no theoretical limits on reading length. Sorghum bicolor (L.) Moench is diploid (2n = 2x = 20) with a genome size of about 730 Mb, and its genome sequence information is released in the Phytozome database. Therefore, sorghum can be used as a good reference. However, plant species have complex and large genomes when compared to animals or microorganisms. As a result, complete genome sequencing is difficult for plant species. MinION sequencing that produces long-reads can be an excellent tool for overcoming the weak assembly of short-reads generated from NGS by minimizing the generation of gaps or covering the repetitive sequence that appears on the plant genome. Here, we conducted the genome sequencing for S. bicolor cv. BTx623 while using the MinION platform and obtained 895,678 reads and 17.9 gigabytes (Gb) (ca. 25× coverage of reference) from long-read sequence data. A total of 6124 contigs (covering 45.9%) were generated from Canu, and a total of 2661 contigs (covering 50%) were generated from Minimap and Miniasm with a Racon through a de novo assembly using two different tools and mapped assembled contigs against the sorghum reference genome. Our results provide an optimal series of long-read sequencing analysis for plant species while using the MinION platform and a clue to determine the total sequencing scale for optimal coverage that is based on various genome sizes.

Impact of Environmental Factors on Stilbene Biosynthesis

“…Remarkable exceptions to this role are sorghum, in whose genome only one STS gene has been identified [74,75], and grapevine, which possesses an uncommonly large number of STS genes. Both grapevine and sorghum genomes have been entirely sequenced [74,76]. Early Southern-blot analysis suggested that the grapevine STS gene family consisted of 15-20 members [77].…”

A Chromosome-Level Genome Assembly of the Pygmy Mole CricketXya riparia

“…A total of 2 μg of genomic DNA was needed for nanopore sequencing ( Lee et al 2019 ). The genomic DNA was prepared using the NEB Next FFPE DNA Repair Mix kit (M6630, USA) and then processed with the ONT Template prep kit (SQK-LSK109, UK) following the manufacturer’s instructions ( Kim et al 2019 ).…”

Cas9-targeted Nanopore sequencing rapidly elucidates the transposition preferences and DNA methylation profiles of mobile elements in plants

“…While whole-genome Nanopore sequencing provides excellent resolution to potentially detect all TEIs in a genome, it is expensive when the genome is relatively large. For example, in our hands and based on previous reports using different species, it is challenging to generate more than 8 Gb of data per single MinION flow cell from plant material (Lee et al, 2019; Schalamun et al, 2019; Dmitriev et al, 2021). Obtaining only 4400 CANS reads (equivalent to 0.2× coverage of the A. thaliana genome) on a single flow cell, we were able to detect 86% of the genetically inherited EVD insertions.…”