Saccharum hybrid cultivar SP80-3280 Genome sequencing using TruSeq Synthetic Long Reads
Sugarcane stalks from genotype SP80-3280 (Saccharum spp.) were gently provided by Centro de Tecnologia Canavieira (CTC), Piracicaba SP, Brazil. Stalks were sectioned in order to contain only one bud and were germinated in trays containing vermiculite. After one month, plants with homogenous phenotype were transferred to pots (3.5 L) containing pine-bark substrate and vermiculite (1:1). Plants were fertilized with N:P:K every 15 days. The pots were watered daily and every week their distribution inside the greenhouse was randomized.  The leaf roll of a two-month old plant was  collected and immediately frozen on liquid nitrogen. Tissue was grounded until fine powder using a mortar and pestle. High molecular weight DNA was extracted using CTAB as follows:  100 mg of fresh frozen tissue was added to  600 µL of heated (65°) extraction buffer corrected to pH 5 (100 mM Tris-HCl 1M pH 8, 20 mM EDTA pH 8; 1,4 M NaCl; 2% CTAB; 1% PVP; 0,1 % ß-mercaptoethanol), incubated for 60 minutes with eventual shaking. One volume of chloroform:isoamyl alcohol (24:1) was added, homogenized  and centrifuged for 10 minutes at 14.000 rpm. The supernatant was transferred to a new tube and 0.7 V of isopropanol  and 1/10 V of sodium acetate (3 M) was added. After overnight incubation (-20°C), the mixture was centrifuged for 10 minutes at 14.000 rpm. The supernatant was discarded and the pellet was washed with ice-cold 70% alcohol.  The DNA was resuspended in 20 µL of TE buffer, treated with RNAse, visualized in 1% agarose gel and quantified in spectrophotometer.    Six µg of DNA were sent to Illumina Inc, CA for DNA sequencing using TruSeq Synthetic long reads technology, through their FastTrack Sequencing Service. Briefly, genomic DNA is sheared into long fragments, ligated to amplification adapters and separated into 384-well plates using limiting dilution, such that each well contain approximately 300 molecules. The fragments in each well are amplified by long-range PCR, and later used to make Nextera libraries. The fragments of different wells will have different barcode indexes, introduced during the library preparation. Fragments are sequenced in an Illumina HiSeq2000 system using paired-end chemistry. Sequenced paired-end reads are demultiplexed and assembled separately by barcode using a proprietary short read assembler [19]. Sample preparation kits are available from Illumina and the assembly software is accessible on BaseSpace using the TruSeq Long-Read Assembly App.