title: Illumina HiSeq 4000 paired end sequencing; Ultradeep sequening data: Clonal evolution of longitudinally sampled triple negative breast cancers using tumour and cell-free genomic sequencing
design: Ultradeep sequening data: Clonal evolution of longitudinally sampled triple negative breast cancers using tumour and cell-free genomic sequencing
library_construction_protocol: Patients with histologically diagnosed breast cancer, progressed under any treatment modality, with a receptor phenotype that was negative for ER, PR, and HER2, and whose sites of progression were amenable to biopsy and who were previously operated upon at Tata Memorial Hospital, were eligible to be consented to participate in this study. We recruited nine patients to this study of whom 3 patients (P2, P4 and P7) could be followed longitudinally during the course of their therapy, while other 6 were lost to follow up after initial tissue bio banking at 1st progression. Patients P2, P4 and P7 were recruited at the time of their first progression post adjuvant chemo and radiotherapy. We bio-banked the tumour tissue from the first progression site in RNA-Later along with 15 ml blood in EDTA tubes.  DNA from fresh-frozen and buffy coat samples was extracted using the Qiagen DNA mini kit (Cat No./ID: 51306). A total of 6 mL of plasma from each patient sample (except for one sample for which 3.6 mL blood was used) was used to extract ctDNA using the Circulating Nucleic Acid kit (Cat No./ID: 55114) from Qiagen® (Venlo, The Netherlands), according to the manufacturer's instructions. Then, 6 mL ctDNA from each sample was eluted into 55 µL of AVE buffer and stored at −20°C. The concentration of the extracted ctDNA was determined using the Qubit dsDNA HS (High Sensitivity) Assay Kit (Invitrogen, Carlsbad, CA, USA), and the size distribution of ctDNA was assessed using Fragment analyzer.  Deep sequencing was performed on selected somatic mutations that were identified using exome sequencing data (E-MTAB-11379). An AmpliSeq custom DNA panel (http://sapac.support.illumina.com/downloads/ampliseq-for-illumina-custom-and-community-panels-reference-guide-1000000036408.html) was designed for library preparation. Targets were amplified using PCR (16 cycles at 99°C for 15 sec and 60°C for 4 min). The amplicons (125 bp/mutation) generated from this step were subjected to partial digestion (using the FuPa reagent), followed by indexing and cleaning using AMPureXP beads. This was followed by one more round of PCR amplification (7 cycles at 98°C for 15 sec and 64°C for 1 min) and subsequent cleaning using AMPure XP beads.