Illumina Genome Analyzer IIx paired end sequencing; "MAGIC.790"
Low-coverage genomic sequencing of 488 Arabidopsis thaliana MAGIC recombinant inbred lines
"Leaves were harvested for DNA extraction. DNA isolation was performed at the John Innes Centre, UK, in 96-well plates using the DNeasy96Plant KitandDNeasy 96Protocol (http://www. quiagen.com). Sequencing was performed by the Oxford Genomics Centre, UK. Samples were quantified using the Quant-iT PicoGreen dsDNA Kits (Invitrogen, Carslbad, CA) and a Genios plate scanner (Tecan, Männedorf, Switzerland) according to manufacturer specifications. Sample integrity was assessed using 1% agarose gel. DNA (~300 ng) was fragmented using a Covaris S2 system with the following settings: Intensity: 5, Duty Cycle: 20, Cycles per Burst: 200, Time: 60 sec. Distribution of fragments after shearing was determined using a Tapestation D1200 system (Agilent/Lab901, Santa Clara, CA).  DNA Libraries were constructed using the NEBNext DNA Sample Prep Master Mix Set 1 Kit (New England Biolabs, Beverly, MA), with minor modifications, and a custom automated protocol on a Biomek FX (Beckman, Fullerton, CA). Ligation of adapters was performed using Illumina Adapters (Multiplexing Sample Preparation Oligonucleotide Kit). Ligated libraries were size selected using Ampure magnetic beads (Agencourt, Beckman, Fullerton, CA). Each library was PCR enriched with 25 mM each of the following custom primers: Multiplex PCR primer 1.0 5prime-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3prime Index primer 5prime-CAAGCAGAAGACGGCATACGAGAT[INDEX]CAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3prime Indexes used were 8 bp long. Enrichment and adapter extension of each preparation was obtained using 5 ml of size-selected library in a 50 ml PCR reaction. After 10 cycles of amplification (cycling conditions as per Illumina recommendations), the reactions were purified with Ampure beads (Agencourt/Beckman). The final size distribution was determined using a Tapestation 1DK system (Agilent/Lab901). The concentrations used to generate the multiplex pool were determined by Picogreen.  The library resulting from the pooling was quantified using the Agilent qPCR Library Quantification Kit and a MX3005P instrument (Agilent) before sequencing on an Illumina GAIIx as 50 or 100 bp paired end reads. All steps for library construction, including the setup of the PCR reaction were performed on a Biomek FX (Beckman). Post PCR cleanup was carried out on a Biomek NXp (Beckman) whereas a Biomek 3000 (Beckman) was used to generate the pools of 96 indexed libraries."