Nextera mate pair library sample prep kit

All library preparation and sequencing was performed at the Roy J. Carver Biotechnology Center at University of Illinois at Urbana-Champaign. Two shotgun libraries (350-450 bp, 500-700 bp) were prepared from the DNA of a single haploid male with the Hyper Kapa Library Preparation kit (Kapa Biosystems). Three mate-pair libraries (3-5 kb, 8-10 kb, 15-20 kb) were constructed from DNA pooled from five individual males using the Nextera Mate Pair Library Sample Prep kit (Illumina, CA), followed by the TruSeq DNA Sample Prep kit. A single RNA library was constructed from pooled RNA from the six female tissue samples with the TruSeq Stranded mRNA Library Construction kit (Illumina, CA).
DNA libraries were quantitated by qPCR and sequenced on a HiSeq2500 for 251 cycles from each end of the fragments using a TruSeq Rapid SBS kit version 2. Shotgun libraries were sequenced on a single lane, and mate-pair libraries were pooled and sequenced on a single lane. RNA libraries were sequenced on a single lane for 161 cycles from each end of the fragments. Fastq files were generated and demultiplexed with the bcl2fastq v1.8.4 Conversion Software (Illumina).

High-molecular-weight DNA was isolated from leaf material of seedlings of ‘Haruna Nijo’²² and size selected for a molecule size of 40 kb or higher. The 440-bp paired-end (PE) libraries were prepared with the Hyper Kapa Library Preparation kit (Kapa Biosystems) with no polymerase chain reaction amplification. The 8- to 10-kb mate-pair (MP) libraries were constructed with the Nextera Mate Pair library Sample Prep kit (Illumina, San Diego, CA, USA) followed by the TruSeq DNA Sample Prep kit. The 10X libraries were constructed with the Chromium Genome Library Kit & Gel Bead Kit v2 (10X Genomics). Sequencing was performed following Sato et al.²¹ In brief, the 440-bp PE libraries were sequenced for 251 cycles using a NovaSeq 6000 system (Illumina). The 10X and MP libraries were sequenced for 151 cycles from each end of the fragments on the NovaSeq 6000 system. All libraries were prepared and sequenced at the University of Illinois Roy J. Carver Biotechnology Center (Urbana, IL, USA). In situ Hi-C libraries were prepared as described by Padmarasu et al.²³ (link) Sequencing data generated from each of the libraries are listed in Supplementary Table S1. The Hi-C data were used to prepare chromosome-scale assemblies using the TRITEX pipeline,¹⁹ (link) which was also used for the contig assembly and scaffolding with the PE, MP, and 10X data (Supplementary Table S1).