Computational protocol: Multivalent Chromosomal Expression of the Clostridium botulinum Serotype A Neurotoxin Heavy-Chain Antigen and the Bacillus anthracis Protective Antigen in Lactobacillus acidophilus

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Protocol publication

[…] The High-Throughput Sequencing and Genotyping Unit of the Roy J. Carver Biotechnology Center, University of Illinois at Urbana-Champaign, performed the preparation of the mRNA libraries and RNA sequencing. Initially, rRNA was removed with the Ribozero Bacteria kit (Illumina, San Diego, CA) followed by library preparation with the TruSeq Stranded RNA Sample Prep kit (Illumina, CA). The libraries were pooled in equimolar concentration, and each pool was quantitated by quantitative PCR (qPCR) and sequenced on one lane for 161 cycles using the Illumina HiSeq 2500 Ultra-High-Throughput Sequencing system (with a read length of 160 nucleotides) with the HiSeq SBS sequencing kit version 4. Fastq files were generated and demultiplexed with the bcl2fastq v2.17.1.14 Conversion Software (Illumina). Adapter sequences were removed, and raw sequences were assessed for quality using Fast QC version 0.11.4 ( Subsequent processes were performed with Geneious 9.0.5 (). Raw reads were trimmed to remove bases with an error probability limit of 0.001 (Phred score of 30), and reads of <20 nucleotides were removed. These sequences were then mapped to the reference genomes (accession numbers NC_006814 and modified NC_006814 for strains NCK2310, NCK2326, and NCK2345) using the Geneious mapper (). Geneious software was used to calculate the normalized transcripts per million (TPM) and to compare expression levels between the control (NCK1909) and integration (NCK2310, NCK2326, and NCK2345) strains. For this study, genes were considered differentially expressed if they had a log2 differential expression value greater than 2 and less than −2 and a differential expression absolute confidence (the negative base 10 log of the P value) value greater than 6. […]

Pipeline specifications

Software tools BCL2FASTQ Conversion Software, FastQC, Geneious
Application RNA-seq analysis
Organisms Clostridium botulinum, Bacillus anthracis, Homo sapiens, Lactobacillus acidophilus, Escherichia coli, Lactobacillus acidophilus NCFM
Chemicals Lactic Acid