Truseq nano dna protocol

DNA was extracted using the Machery-Nagel Nucleospin Plant II Kit at the Australian Genome Research Facility (AGRF, Adelaide, Australia). The extracted DNA was then sonicated for random sheering and Illumina’s TruSeq Nano DNA protocol was used for size selection and sequencing adapter and barcode ligation. The hybrid-capture enrichment reactions were carried out following the MyBaits protocol v.2 (www.mycroarray.com/pdf/MYbaits-manual-v2.pdf) using the high stringency wash buffer and 12 cycles of post-capture PCR. Following capture 100 bp paired-end sequencing with dual indexing of 89 samples was performed on one lane of an Illumina HiSeq 2000 at AGRF (Melbourne, Australia). Sequence data was subsequently processed using the Illumina CASAVA pipeline (version 1.8.2).

Fifty-seven bacterial strains isolated between 1963 and 2008, mainly from Asia (Supplementary Data S1) and stored as lyophiles at −80 °C, were chosen to complement the collection of available modern genomes. Strains were grown at 28 °C on YPGA (7 g L⁻¹ yeast extract, 7 g L⁻¹ peptone, 7 g L⁻¹ glucose, 18 g L⁻¹ agar, supplemented by 20 mg L⁻¹ propiconazole, pH 7.2). Single cultures were used for DNA extraction using the Wizard® genomic DNA purification kit (Promega) following the manufacturer’s instructions. Quality assessment was realized for concentration using QuBit (Invitrogen) and Nanodrop (Thermo Fisher Scientific) fluorometers. Library preparation of the modern strains was outsourced to Fasteris where classic TruSeq Nano DNA protocol following Nextera enzymatic DNA fragmentation was applied (Illumina). Sequencing for both historical and modern DNA was performed in a paired-end 2 × 150 cycles configuration on a NextSeq500 machine in several batches, with samples from both types of libraries being independently treated.

For NGS sequencing of the murine Trp53 locus, DNA was isolated from enlarged spleens of chemotherapy-naive TC mice and animals that had relapsed after four cycles of cyclophosphamide treatment. Trp53 exons covering the DNA-binding domain (residues 90-300) were amplified by PCR (Phusion Taq polymerase, NEB, primer sequences are listed in Supplementary Table 4). An amount of 100 ng of purified PCR products was used as template for the indexed library preparation. We followed the Illumina TruSeq Nano DNA protocol, skipping the fragmentation step. After library validation and quantification (Agilent 2100 Bioanalyzer), equimolar amounts of library were pooled. Pools of 12 libraries were quantified by using the Peqlab KAPA Library Quantification Kit and the Applied Biosystems 7900HT Sequence Detection System. Pools were sequenced using an Illumina HiSeq 4000 sequencer with a paired-end (76 × 7 × 76 cycles) protocol generating ~700 k reads/sample. The reads were aligned to the mouse GRCm38 reference with the Burrows-Wheeler Aligner (BWA 0.7.15) and the variants were called with the Genome Analysis ToolKit (GATK 3.7 Unified Genotyper) and annotated with Annovar (version 2016-02-01).