Computational protocol: Transcriptional and Linkage Analyses Identify Loci that Mediate the Differential Macrophage Response to Inflammatory Stimuli and Infection

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Protocol publication

[…] Reads were initially mapped to the mouse genome (mm9) and the Toxoplasma (ME49 v8.2) genome using Bowtie (2.0.2) [] and Tophat (v2.0.4) []. We then estimated gene expression levels in cufflinks (v2.0.0) [] using the Illumina iGenomes refseq genome annotation (NCBI build 37.2) with the multi-read, compatible-hits corrections and upper quantile normalization options enabled. Because the reference genome to which we mapped the RNA-seq reads is based on the C57BL/6J genomic sequence, and due to the known polymorphisms between the AJ and C57BL/6J, we suspected that biases introduced at the read mapping stage might affect our expression results. To mitigate this potential bias towards the reference allele, we created a copy of the mouse genome in which all the known single nucleotide polymorphisms (SNPs) between AJ and B6, as annotated by the Wellcome Trust Sanger Institute sequencing (ftp://ftp-mouse.sanger.ac.uk/current_snps/), were converted to a third (neutral) nucleotide that is different from both the reference and AJ allele []. However, this did not substantially change the average proportion of uniquely mapped reads or expression profiles of individual genes in all the samples. In the end we used the mapping data generated from the synthetic genome to quantify gene expression levels. […]

Pipeline specifications

Software tools Bowtie, TopHat, Cufflinks
Databases iGenomes
Application RNA-seq analysis
Organisms Mus musculus, Toxoplasma gondii, Toxoplasma
Diseases Communicable Diseases, Neoplasms
Chemicals Nitric Oxide