Computational protocol: Oxygen-limited metabolism in the methanotroph Methylomicrobium buryatense 5GB1C

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Protocol publication

[…] RNA was extracted as previously described (). RNA sequencing was performed by GENEWIZ (South Plainfield, NJ, USA) using Illumina HiSeq2500 1 × 50 (single ended) reads. The raw reads from the sequencing facility were aligned to the annotated M. buryatense 5G genome as downloaded from JGI’s IMG on July 14, 2017 (). Alignment was performed using BWA version 0.7.12-r1044 using the BWA-MEM algorithm and default parameters (). The alignments were post-processed into sorted BAM files with SAMTools version 1.2-232-g87cdc4a (). Reads were attributed to open reading frames (ORFs) using the htseq-count tool from the ‘HTSeq’ framework version 0.8.0 in the ‘intersection-nonempty’ mode (). Differential abundance analysis was performed with DESeq2 1.2.10 (; ) using R 3.3.1.Genes were considered to be differentially expressed if there was an average change of greater than 2-fold when comparing normalized counts as well as an adjusted p-value of less than 1E − 05 (). […]

Pipeline specifications

Software tools BWA, SAMtools, HTSeq, DESeq2
Application RNA-seq analysis
Chemicals Carbon, Methane, Oxygen, Superoxides, Lactic Acid, Succinic Acid