Summary of whole genome sequencing for B. abortus
datasetposted on 29.08.2019, 15:15 by Xavier De BolleXavier De Bolle
Two liquid cultures of WT B. abortus were inoculated in 2YT rich medium from the same plate. One of them was divided in 5 subcultures and diluted (1:10) in liquid cultures twice every 24 h, before to be plated. The remaining original liquid culture was used to infect five C57BL/6 mice and RAW 264.7 macrophages. Mice were injected intraperitoneally as described above and were euthanized at 60 h post inoculation to recover spleens. RAW 264.7 macrophages were infected as described above and bacteria were plated at 6 and 48 h post infection. Five streaks were made from five isolated colonies obtained after passage in either liquid cultures, mice or RAW 264.7 macrophages from different wells. The five streaks served for inoculation of liquid cultures, from which genomic DNA was extracted (NucleoSpin Tissue extraction kit, Macherey-Nagel). Samples were sequenced with Illumina sequencing technique using NextSeq500 run Mid PE150 after preparing a TruSeq DNA library (performed by Genomics Core Leuven, Belgium). Sequencing hits were mapped on the genome of B. abortus 544 and, for each colony, all the nucleotides that were different from the reference strain were compiled in an Excel table (performed by Genomics Core Leuven, Belgium) made available here. Mutations were counted for each isolated colony by excluding regions corresponding to microsatellites and with less than 10 reads. The exclusion procedure is shown in the different tabs of the Excel file.