Gossypium hirsutum (AD1) 'Bar32' genome NSF_v1

Acid-delinted seeds of BARBREN-713 and BAR 32-30 (G. hirsutum) were chipped and rolled in moist germination towels. Rolled towels were placed in an incubator set at 30C for 4 days to germinate seeds. DNA was extracted from resulting seedlings using the Qiagen Genomic Tip kit (Qiagen, Hilden, Germany). The sequencing libraries were constructed at the Brigham Young University DNA Sequencing Center (DNASC). DNA shearing of both libraries was done on a Megaruptor2 (~20 kb) (Diagenonde Inc., Denville, NJ, USA). HMW DNA was partitioned into 13 bins using the Sage Elf (Sage Science, Beverly, MA, USA), and the top 5 bins were run on a Fragment Analyzer (Agilent Technologies, Santa Clara, CA, USA) to select the appropriate bin size range (15–18 kb). Libraries were made using the SMRTbell Express Template Prep kit as recommended by Pacific Biosciences (PacBio; Menlo Park, CA, USA). Five PacBio cells were sequenced from each library on the Pacific Biosciences Sequel 2 system. The PacBio reads were assembled using hifiasm (Cheng et al. 2021) and default parameters. Both assembled genomes were aligned to previously assembled genomes of G. hirsutum using minimap2 (Li 2018; Chen et al. 2020) and visualized by dotPlotly (https://github.com/tpoorten/dotPlotly, last accessed 8/17/21). Manual scaffolding was used to create pseudomolecules of both genomes.

Hi-C libraries were constructed from the same seedling tissue using the Plant Hi-C Kit (Phase Genomics, Seattle, WA, USA). Short-read sequencing (Illumina, San Diego, CA, USA; 150PE) of the libraries was performed by BGI Americas Corp (Cambridge, MA, USA). The Hi-C data of both genomes were mapped to their respective assembled genome sequence using bwa mem (Li and Durbin 2009). The Hi-C interactions were used as evidence for contig proximity and in scaffolding contig sequences. Within their respective set of mapped reads, matlock (https://github. com/phasegenomics/matlock, last accessed 8/17/21) was used to identify linkages between different genomic regions in the bam file. Juicebox (Robinson et al. 2018) was used to visualize the linkages along the pseudomolecules.

Table 1. The assembled genomes of BARBREN-713, BAR 32-30, and TM-1 (Chen et al. 2020)

  ^a Contig metrics reports are from the raw output file of hifiasm.
  ^b Scaffold metrics reported are after manual scaffolding.

 Publication: Perkin et al., Genome assembly of two nematode-resistant cotton lines (Gossypium hirsutum L.). G3 5 August 2021.