title: Illumina Genome Analyzer IIx paired end sequencing; Whole exome sequencing of Cervical Squamous samples
design: Whole exome sequencing of Cervical Squamous samples
library_construction_protocol: Ten pretreatment cervical tumour biopsies along with the matched blood samples from Indian patients with advanced stage cervical cancer (FIGO stage IIIB) were taken for the study. A generic consent for basic research was obtained prior to obtaining the biopsies. Since this was a retrospective study, patient informed consent for this particular project could not be obtained and permission for waiver from Institute Ethics Committee (IEC) was obtained. The study was approved by IEC. All procedures pertaining to sample collection and processing were in conformity with the IEC. The biopsies were obtained and stored in liquid Nitrogen as described earlier [8], from histologically proven, primary cervical tumour, before the start of radical radiation therapy. The samples were coded for identification by the physician prior to testing and were assigned a laboratory code to maintain confidentiality. Buffy coat was separated from blood samples by centrifugation. Cryosectioning of the cervical biopsies was done for hematoxylin-eosin (H&E) staining as well for isolation of DNA. The H&E stained sections were examined by the pathologist to assess the percentage of tumour in the sample. Samples with >70% tumour were taken for whole exome sequencing study. Genomic DNA was isolated from the tumour sections and buffy coat from matched blood samples, using DNA Mini kit (Qiagen, Hilden, Germany) according to the manufacturerâ€™s instructions. RNase treatment of the DNA samples was done using RNase A (Sigma, St. Louis, MO, USA) Matched tumour and blood samples from 10 cervical cancer patients were subjected to exome sequencing using Illumina platform. Exome capture and paired end sequencing of DNA was done using Paired-End Sample Preparation kit (Illumina, San Diego, CA, USA) followed by in-solution capture of genomic DNA using SureSelect Human All Exon kit (Agilent Technologies, Santa Clara CA, USA), targeting 37 Mb sequence from exons.