Computational protocol: In Vitro Culture of the Insect Endosymbiont Spiroplasma poulsonii Highlights Bacterial Genes Involved in Host-Symbiont Interaction

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[…] S. poulsonii DNA was extracted from fly hemolymph as previously described (). Processing of the samples was performed in the University of Lausanne Genomic Technologies Facility. The DNA was sheared in a Covaris g-TUBE (Covaris, Woburn, MA, USA) to obtain 20-kb fragments. After the DNA was sheared, the size distribution of the DNA fragments was checked on a Fragment Analyzer (Advanced Analytical Technologies, Ames, IA, USA). Five micrograms of the sheared DNA was used to prepare an SMRTbell library with the PacBio SMRTbell template prep kit 1 (Pacific Biosciences, Menlo Park, CA, USA) according to the manufacturer’s recommendations. The resulting library was size selected on a BluePippin system (Sage Science, Inc., Beverly, MA, USA) for molecules larger than 20 kb. The recovered library was sequenced on one SMRT cell with P6/C4 chemistry and MagBeads on a PacBio RSII system (Pacific Biosciences, Menlo Park, CA, USA) in a 240-min movie. Assembly was performed with HGAP (hierarchical genome assembly process) version 2 from the PacBio smrtpipe (v2.3.0). Circularization of main contig 1 was performed using Amos (v3.1.0; Amos Consortium; http://amos.sourceforge.net). Plasmid contig 7 was refined using the PacBio read data from Paredes et al. () with Quiver version 1. Genome annotation was performed with Prokka v 1.11 () using parameters --addgenes --genus Spiroplasma --species poulsonii --gcode 4 --rawproduct –rfam --rnammer). The Nucmer tool (Mummer suite v3.23 []) was used to align coding sequences (CDS) from the annotated version (accession no. JTLV01000000) to the new annotation. Some gene product annotations were refined using NCBI PSI-blast tool. Multiple alignments of Spiroplasma plasmids have been performed using MAUVE version 2.4.0 (). [...] RNA was extracted from (i) the hemolymph of 30 1-week-old infected flies and (ii) 20 ml of 4-month-old in vitro culture pelleted by 30 min of centrifugation at 16,000 ×  g by the TRIzol method following the manufacturer’s instructions. Three independent replicates were prepared for each condition. Libraries were prepared using the Illumina Truseq RNA kit and sequenced on an Illumina HiSeq 2000 system at the University of Lausanne Genomic Technologies Facility. Purity-filtered reads were adapters and quality trimmed with Cutadapt v.1.8 (). Reads matching to rRNA sequences were removed with fastq_screen v. 0.9.3 (Babraham Bioinformatics; http://www.bioinformatics.babraham.ac.uk/projects/fastq_screen/). Remaining reads were further filtered for low complexity with reaper v. 15-065 (). Reads were aligned against the Spiroplasma poulsonii MSRO (v2) genome using STAR v. 2.5.2b (). The number of read counts per gene locus was summarized with htseq-count v. 0.6.1 () using Spiroplasma poulsonii MSRO (v2) gene annotation. The quality of the transcriptome sequencing (RNA-seq) data alignment was assessed using RSeQC v. 2.3.7 (). Statistical analysis was performed for genes in R version 3.4.1 (). Genes with low counts were filtered out according to the rule of one count per million (cpm) in at least one sample. rRNA and tRNA gene counts were discarded. Library sizes were scaled using TMM normalization with EdgeR package version 3.18.1 () and log cpm transformed with limma voom function, limma package version 3.32.5 (). Differential expression was computed with limma () by fitting the samples into a linear model and performing “in fly” versus “in culture” comparison. Moderated t test was used, and adjusted P values were computed by the Benjamini-Hochberg method, controlling for the false-discovery rate. […]

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