Computational protocol: Anaerobically Grown Escherichia coli Has an Enhanced Mutation Rate and Distinct Mutational Spectra

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Protocol publication

[…] The E. coli REL4536 reference genome was manually annotated using information provided by Barrick et al. [] and the E. coli REL606 GenBank file (NCBI Reference Sequence NC_012967.1). Sequence data from BGI had been filtered to remove reads containing ≥ 10% unreadable bases, ≥ 20% low quality (≤ Q20) bases, adapter contamination or duplicate read-pairs and FastQC [] was used to check the quality of the sequence data before analysis. BPSs, indels and MGE movement were identified using breseq [, ] with default parameters. To detect SVs, genomes were assembled de novo using SPAdes 3.0.0 [] with default parameters. Assemblies were aligned to the reference E. coli REL4536 genome using the NUCmer pipeline in MUMmer [] and Mauve [] with default parameters to identify SV breakpoints. Verification of SV breakpoints was carried out using PCR using primers in . PCR (25 μL total reaction volume) was performed using 1× reaction buffer (Invitrogen), 2 mM Mg+2 (Invitrogen), 0.2 mM deoxyribonucleotide triphosphate mixture (Invitrogen), 0.2 μM forward primer, 0.2 μM reverse primer, 0.02 unit/μL of Platinum High Fidelity Taq DNA polymerase (Invitrogen) and 1–100 ng of genomic DNA. PCR amplifications were performed on Eppendorf proS Mastercycler PCR machines and a standard PCR programme was used: initial denaturation at 94°C for 3 min, followed by 30 cycles of 94°C for 30 sec, annealing temperature (primer dependent) for 30 sec and extension at 68°C for 1 min per kb of product. The final elongation was for 10 min at 68°C. Using UltraPure Agarose (Invitrogen), 1% (w/v) gels were made up in 1× Tris-acetate-EDTA (TAE) buffer containing 1× SYBR safe nucleic acid dye (Life Technologies) to separate and visualize the PCR products.In this study, an increase in IS element copy number, as well as a change in the location of an IS element, were both counted as IS insertions. Spontaneous mutation rates for aerobically and anaerobically grown E. coli lineages were calculated with the equation μ = m/(L*N*T), where μ is the mutation rate per genome per generation, m is the total number of observed mutations, L is the number of MA lineages, N is the number of generations between bottlenecks and T is the number of bottlenecks (180 for aerobic lineages and 144 for anaerobic lineages). To obtain the mutation rate per nucleotide per generation, the equation μ/G was used, where G is the size of the genome that was sequenced (). To obtain the mutation rate per genome per day, the equation μ' = m/(L*D) was used, where μ' is the mutation rate per genome per day, m is the total number of observed mutations, L is the number of MA lineages and D is the number of days of evolution (180 for aerobic lineages and 432 for anaerobic lineages). [...] The transcriptomes of three aerobic and two anaerobic stationary phase E. coli REL4536 cultures were sequenced. Aerobic cultures were grown in 250 mL serum bottles for 9 h while anaerobic cultures were grown for 16 h. All cultures were grown in 50 mL DM25 aliquots at 37°C. Cells were harvested by centrifugation at 8,000 g for 10 min at 4°C. Media was discarded and cell pellets were snap-frozen in liquid nitrogen. RNA was extracted from cultures using a hot lysis buffer and acid phenol-based extraction method [] and isopropanol precipitation. Samples were lysed with 5 mL of 2% SDS solution at 65°C and poured into 10 mL of phenol:chloroform:isoamyl alcohol (125:24:1, v:v:v, pH 4.5; Ambion) and incubated at 65°C for 15 min. Following RNA extraction, Turbo DNase (Ambion) was used to treat the samples as per manufacturer’s instructions. Each sample was split into five 20 μL aliquots and two rounds of DNase treatment were performed on each aliquot. Following DNase treatment, RNA samples were purified using the Qiagen RNeasy Mini kit as per the manufacturer’s instructions. For each sample, the DNase-treated aliquots were pooled together before purification. RNA quality was measured using the Agilent 2100 Bioanalyzer (Agilent Technologies) with the RNA 6000 Nano Chip kit according to the manufacturer’s instructions while RNA was quantified using the Quant-iT RNA Assay kit (Life Technologies) and measured on a Qubit 2.0 fluorometer.Total RNA libraries of 200 bp inserts were sequenced by Illumina HiSeq 2000 at BGI. Paired-end sequencing with 90 bp reads was performed. The quality of reads was assessed by using FastQC [] and any reads with a quality score ≤ Q28 were trimmed using Trim Galore![]. Bowtie 2 [] was used with default parameters, to remove any sequence reads aligning to ribosomal RNA, transfer RNA and non-coding RNA sequences. EDGE-pro (Estimated Degree of Gene Expression in PROkaryotes) [] and DESeq2 [] were used on the remaining reads to identify differential expression between aerobic and anaerobic environments. The RNA sequence data is available at NCBI GEO accession GSE80451. […]

Pipeline specifications

Software tools FastQC, Trim Galore!, Bowtie, EDGE-Pro, DESeq2
Application Non-coding RNA analysis
Organisms Escherichia coli
Chemicals Oxygen