Babesia bovis RNA-Seq from blood and tick stages
Babesia bovis is a hemoprotozoan parasite of cattle that has a complex life cycle within vertebrate and invertebrate hosts. In the mammalian host, B. bovis undergoes asexual reproduction while in the tick midgut, gametes are induced, fuse, and form zygotes. The zygote infects tick gut epithelial cells and transform into kinetes that are released into the hemolymph and invade other tick tissues such as the ovaries, resulting in transovarial transmission to tick offspring. To compare gene regulation between different B. bovis life stages, we collected parasites infecting bovine erythrocytes and tick hemolymph. Total RNA samples were isolated, and multiplexed libraries sequenced using paired-end 100 cycle reads of a HiSeq 2500. The data was normalized using the TMM method and analysed for significant differential expression using the generalized linear model likelihood ratio test (GLM LRT) in edgeR.
Steps to reproduce
Three splenectomized Holstein calves approximately four months of age and determined to be Babesia-free were used for acquisition of B. bovis Texas strain by Rhipicephalus microplus, La Minita strain. R. microplus larvae were placed under a cloth patch on calves. When ~1% of the ticks had molted to the adult stage, calves were intravenously inoculated with B. bovis infected erythrocytes to synchronize female tick repletion with an ascending parasitemia. Replete female ticks were collected and incubated at 26°C in 96% relative humidity to allow B. bovis development. Blood samples from an acute parasitemia were cultured for 5 days at 3% oxygen and 5% carbon dioxide. Cultures were centrifuged, media removed, cells suspended in TRIzol and stored at -80°C. Babesia bovis kinetes were collected from replete female ticks by extraction using pressurized capillary tubing, pooled, and stored in TRIzol at -80°C. RNA samples were isolated and treated with TURBO DNase. Sample RNA concentrations were determined using a NanoDrop 1000 and tested for residual DNA. Total RNA was monitored for quality control using the Agilent Bioanalyzer Nano RNA chip and NanoDrop absorbance ratios for 260/280nm and 260/230nm. Library construction was performed according to the Illumina TruSeq mRNA stranded protocol. Using an input quantity for total RNA within the recommended range, mRNA was enriched using oligo dT magnetic beads. The enriched mRNA was chemically fragmented. First strand synthesis used random primers and reverse transcriptase to make cDNA. After second strand synthesis the ds cDNA was cleaned using AMPure XP beads and the cDNA was end repaired and the 3’ ends adenylated. Illumina barcoded adapters were ligated to the ends and the adapter ligated fragments were enriched by nine cycles of PCR. The resulting libraries were validated by qPCR and sized using an Agilent Bioanalyzer DNA high sensitivity chip. The library concentrations were normalized and then multiplexed. The multiplexed libraries were sequenced using paired end 100 cycle chemistry for the HiSeq 2500. The version of HiSeq control software was HCS 2.2.58 with real time analysis software, RTA 1.18.64. Low quality reads were filtered before alignment to the new reference genome using STAR v2.5.2a (2-pass mapping). Counts were generated from alignments for each gene using the Subread feature of Counts v1.6.0. Genes without at least 1 read per million mapped reads across all three samples within a group were removed, data were normalized using the TMM method, and analyzed for differential expression significance testing using the generalized linear model likelihood ratio test (GLM LRT) method in edgeR v3.20.9. The false discovery rate (FDR) method was employed to correct for multiple testing and genes were termed differentially expressed if their log Fold Change (logFC) value was greater than or equal to 1 with the FDR set to 5%.