Nextera mate pair sample preparation kit

The quality of the DNA was assessed by gel-electrophoresis and the quantity was estimated using the Quant-iT PicoGreen dsDNA kit (Invitrogen) and the Tecan Infinite 200 Microplate Reader (Tecan Deutschland GmbH, Mainz, Germany). To obtain the complete genome sequence, a whole-genome-shotgun PCRfree (Nextera DNA Sample Prep Kit; Illumina, Munich, Germany) and an 8 K mate pair library (Nextera Mate Pair Sample Preparation Kit; Illumina) were generated based on the manufacturer’s protocol. After sequencing and processing of the raw data, a de novo assembly was performed using the GS De Novo Assembler software release version 2.8 (Roche, Mannheim, Germany) with default settings. In our approach, we used a 2 x 300 bp paired end sequencing run. For quality-control and filtering, a pipeline including trimmomatic (Bolger et al. [45 (link)]), r2cat [46 (link)] and contig-length vs. read-count analysis [47, 48] was implemented. The resulting data set was manually inspected and improved.

A Chinese Spring wheat BAC library [13 ] was screened at the INRA-CNRGV [44 ] using the primers described in Additional file 6. In a first screening round, primers for the markers CD900298 and wsnp_Ex_c66324_64493429 were used and three BACs were identified (1816F24, 1836C17, 0251E06). A second round of screening was done by using a new set of primers for PM19-A1, which allowed us to identify four additional BACs of interest (1964H07, 0404N02, 1824C13, 1758D08). BAC DNA, isolated using the PhasePrep BAC DNA kit (Sigma-Aldrich, Sydney, Australia), was used in the preparation of paired-end (PE) and mate-pair (MP) libraries. For the paired-end libraries a single library for each BAC clone was prepared using the Nextera XT DNA library preparation kit (Illumina) following the manufacturer’s instructions. For the mate-pair libraries duplicate libraries were prepared on a pool of all BAC DNA using the Nextera Mate Pair Sample Preparation Kit (Illumina) with a lower size exclusion performed with Solid Phase Reversible Immobilization (SPRI) size selection kit (Beckman Coulter, NSW, Australia) to fragments greater than 1500 bp. All libraries were individually barcoded and sequenced on an Illumina HiSeq 2000 using v3 chemistry and Illumina MiSeq using v3 chemistry.

Libraries were prepared from 1 microgram of high molecular weight genomic DNA using Illumina's Nextera Mate Pair Sample Preparation Kit, according to the manufacturer’s instructions for a gel-free preparation of 2 kb effective insert size library (size distribution mode 2 kb). The libraries were sequenced on an Illumina HiSeq 2500 sequencer, 2x100 bp to an average raw coverage depth of 5x. Raw sequence reads were base-called using CASAVA RTA 1.18.

A sibling inbred line, Sp75, was grown under standard greenhouse conditions with a 16-h light (27 °C) and 8-h dark (19 °C) cycle. Young leaves from 20-days-old plants were collected, immediately frozen in liquid nitrogen and stored at −80 °C. DNA was extracted using the QIAGEN DNeasy Plant Mini Kit following the manufacturer's instructions (QIAGEN, Valencia, CA, USA). DNA quality was evaluate via agarose gel electrophoresis and its quantity was determined on a NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA). Paired-end libraries with insert sizes of 150 bp, 200 bp, 300 bp, 500 bp and 1 kb and mate-pair libraries with insert sizes of 3, 10 and 15 kb were constructed using the Genomic DNA Sample Prep kit and the Nextera Mate Pair Sample Preparation kit (Illumina, San Diego, CA, USA), respectively, following the manufacturer's instructions, and sequenced on an Illumina HiSeq 2500 platform with paired-end mode.

The high throughput genomic mate pair libraries from HMCLs were prepared by the Mayo Clinic Genome Core Facilities using the Illumina Mate Pair protocols and their available kit (Nextera Mate Pair Sample Preparation kit, Illumina)^{10 (link),24 (link)}, Then, two samples were run on one lane of an Illumina HiSeq2000 with 50 bp reads. The sequences were aligned to hg19 using BWA, and a BAM file containing only the clustered discordant reads with clustered mates was created. Breakpoints in the IRF4 locus with discordant reads were identified in the BAM file by visual inspection.

The de novo sequencing of the SEMIA3007 genome used a combined strategy involving Illumina – HiscanSQ. The libraries were constructed using a TruSeq® DNA Sample Prep kit and Nextera Mate Pair Sample Preparation kit (Illumina®). The cluster formation of library templates was performed with the TruSeq PE Cluster kit v3 (Illumina®) and the Illumina cBot workstation using conditions recommended by the manufacturer. Paired end 100 base pair (2x100bp) sequencing by synthesis was performed with TruSeq SBS kit v3 (Illumina®) on an Illumina HiscanSQ using protocols defined by the manufacturer. The base call conversion to sequence reads was performed using CASAVA 1.8.3 (Illumina®). As a result, paired-end and mate pair fastq files were trimmed using Scythe 0.991 (https://github.com/vsbuffalo/scythe), Cutadapt 1.7.1 [40 (link)] and the quality of data was filtered by Prinseq program [41 (link)] with Phred ≥20. The sequence assembly was performed using the Spades 3.6.1 program [42 (link)]. The prediction of ORFs and annotation were performed using the Rast system [43 (link)].

Genomic DNA was isolated from Vigna umbellata using an Xcelgen CP Plant DNA isolation kit, and libraries were prepared using an Illumina TruSeq Nano DNA HT library preparation kit for insert sizes of 350 and 550 bp. The mean sizes of the libraries were 664 and 438 bp for the 350-bp insert size and 876 bp for the 550-bp insert size. The library with an insert size of 350 bp was sequenced on the Illumina platform with 2 × 150 bp chemistry to generate ∼30 GB of data, whereas the library with an insert size of 550 bp was sequenced with 2 × 250 bp chemistry to generate ∼15 GB of data. The mate-pair library was prepared from a rice bean sample using an Illumina Nextera Mate-pair Sample Preparation Kit. The mean size of the library was 580 bp. The library was sequenced (2 × 150 bp chemistry) to generate ∼10 GB of mate-pair data. The Pac-Bio library was prepared and sequenced using the PacBio sequencing platform to generate 6.5 GB of data.