Streptomyces SAI-25 mate pair end WGS raw reads
SAI-25 was inoculated in starch casein broth and incubated for 120 h at 28 C. At the end of incubation, the culture was centrifuged at 10,000g for 10 min at 4 C and the cells were washed twice with STE buffer (sucrose 0.3 M, Tris/ HCl 25 mM and Na2EDTA 25 mM, pH 8.0). The supernatant was discarded but the pellet (1 g) was re-suspended in 8.55 ml STE buffer and 950 l lysozyme (20 mg/ml STE buffer) and incubated for 30 min at 30 C. This was followed by the addition of 500 l of SDS (10%; w/v) and 50 l of protease (20 mg/ml) and the mixture was held at 37 C for one h. At the end of incubation, 1.8 ml of NaCl (5 M) was added with gentle mixing to avoid shearing the DNA, and 1.5 ml of CTAB (10%; w/v) in 0.7 M NaCl (CTAB/NaCl solution) and incubated for 20 min at 65 C. Once CTAB was added, all the remaining steps were carried out at room temperature. The lysate was extracted twice with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1; by vol) and centrifuged at 13,000g for 10 min. Finally, the aqueous phase was extracted with chloroform/isoamyl alcohol (24:1, by vol) and transferred to a tube followed by the addition of 600 l of propan-2-ol and DNA spooled out after 10 min. Alternatively, it was recovered by centrifugation at 12,000g for 10 min. The pellet was washed twice with ethanol (70%; v/v), vacuum dried, and dissolved in 2 ml of TE buffer (10 mM Tris/HCl and 1 mM EDTA, pH 8.0). RNase A (50 mg/ml) was added with incubation at 37 C for 2 h. The sample was again extracted with a phenol as described above. DNA was re-precipitated from the aqueous phase with the addition of 100 l of 3 M sodium acetate (pH 5.3) and 600 l of propan-2-ol. The DNA pellet was washed with ethanol (70%; v/v), dried, and dissolved in TE buffer. The purity of the SAI-25 DNA was checked in the agarose gel electrophoresis and quantified using NanoDrop. Briefly, about~5 g high-quality genomic DNA (free from any contaminant and having an A260/280 ratio in the range of~1.8 to 2.0 with DNA concentration100 ng/l) was sent to AgriGenome Labs (Kochi, India) for library preparation and next-generation sequencing using the Illumina platform. The Genomic DNA was fragmented, and a paired-end library with insert size 300 bp, and a mate-pair library with insert size 5 Kbp were prepared. The DNA fragment libraries were validated by tapestation, and sequenced~9.905 million paired-end (100 bp  2) and~6.242 million mate pair (250 bp  2) read on the Illumina HiSeq 2500.