Whole genome sequencing of Acanthamoeba isolated from corneal scraping
The library preparation was carried out at Genotypic Technologys NGS facility by Illumina- compatible NEXTflex Rapid DNA sequencing Bundle (BIOO Scientific, Inc. U.S.A.) librarypreparation kit, by following manufacturers instructions. Briefly, 250ng of Qubit quantified DNA was sheared using Covaris S220 sonicator (Covaris, Inc. USA) to generate specific fragments in the size range 200-300 bp. The sheared fragments werebead-purified and the purified fragments were end-repaired, adenylated and ligated to Illuminamultiplex barcode adaptors as per NEXTflex RapidDNA sequencing bundle kit protocol. Adapter-ligated DNA was further bead-purified and the resultant fragments were amplified for 4cycles of PCR using Illumina-compatible primers provided in the NEXTflex Rapid DNAsequencing Bundle. Final PCR product were bead-purified and assayed for quantity and quality. Quantification of the library was assessed using Qubit flourometer. (Thermo Fisher Scientific, MA, USA) and the fragment size distributions were analyzed on Agilent 2200 TapeStation. Sample was paired-end sequenced on Illumina HiSeq X Ten sequencer (Illumina, San Diego, USA) for 150 cycles. The run set-up was carried out following manufacturers instructions. Afterthe completion of sequencing run, the data was demultiplexed using bcl2fastq software v2.20and Fast Q files were generated based on the unique dual barcode sequences. The sequencing quality was assessed using Fast QC v0.11.8 software. The adapter sequenceswere trimmed and bases above Q30 were considered and low quality bases were filtered offduring read pre-processing and used for downstream analysis