Genomic DNA of five independent mutant strains and one control E. coli ΔtolC strain was sequenced using Illumina Paired-End technology on the MiSeq instrument at the Helmholtz Centre for Infection Research (Braunschweig, Germany). Characteristics of the obtained raw-data sequencing reads are shown in Supplementary Table 1 . Raw data were imported into the Geneious 9.1.337 (link) software package and trimmed of low-quality parts with an error probability threshold of 0.05. It was aligned against the E. coli ΔtolC reference sequence using the ‘Low sensitivity’ option of the Geneious ‘Map to reference…’ sequence aligner. This produced assembly files in which whole genome mean sequencing coverages varied in the range of 108–116. Assembly files were then converted to consensus sequences by the ‘Generate consensus sequence…’ option in the Geneious software and ‘Highest quality’ consensus calling option. Six resulting consensus sequences were aligned to each other and to the reference genome sequence by the ‘progressiveMauve’ algorithm of the MAUVE whole-genome sequence alignment tool38 (link). The variant calling was done by comparing the consensus sequences of each mutant sample against the control E. coli ΔtolC WT consensus sequence. The complete list of mutations is shown in Supplementary Table 2 .
Paired end technology
Manufactured by Illumina
Sourced in Germany
Paired-end technology is a sequencing method used to generate two reads from each DNA fragment, one from each end of the fragment. This technique provides information about the relative orientation and distance between the two reads, which can be used to improve genome assembly and the identification of structural variations in the DNA.
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4 protocols using paired end technology
1
Whole-Genome Sequencing of E. coli Mutants
2
Euplotes focardii Genome Sequencing
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DNA was purified from Euplotes focardii cultures as previously described [25 (link)]. Sequencing was performed by Illumina paired-end technology (a total of 43,588,788 reads covering 4,402,467,588 bp, with an average read length of 100 bp), in collaboration with Dr. Vadim Gladishev’s research group (Brigham and Women’s Hospital and Harvard Medical School, Boston). The sequences were assembled using Newbler.
3
Liver Transcriptome Analysis of Alb-R26^Met and R26^stopMet Mice
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Liver samples from Alb-R26Met and R26stopMet mice were submitted to GATC Biotech for RNA sequencing with Illumina paired-end technology (n = 4 R26stopMet and 3 Alb-R26Met) following the methodology previously reported [12 (link)]. The differential expression of genes was calculated as log2FC, and their significance was assessed.
4
Sequencing of Hot and Cold Evolved Drosophila
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We previously sequenced the hot evolved populations until generation 60 and the cold evolved populations until generation 40. All populations were sequenced as pools (Pool-Seq Schlötterer et al. 2014 (link)) at each 10th generation using the Illumina paired-end technology (Accession numbers PRJEB20533 and PRJEB20780).
Here we additionally sequenced the cold evolved populations at each 10th generation from generation 50 to 100. For each sample the DNA of pooled female and male flies was extracted using a high salt extraction protocol (Miller et al. 1988 (link)) and sheared with a Covaris S2 device (Covaris, Inc. Woburn, MA, USA). Libraries were prepared using the TruSeq DNA PCR-Free protocol (Illumina, San Diego, CA) and sequencing was performed with the Illumina HiSeq X Ten platform (Illumina, San Diego, CA). For an overview of the sequencing data used in this work seesupplementary table S1, Supplementary Material online.
Here we additionally sequenced the cold evolved populations at each 10th generation from generation 50 to 100. For each sample the DNA of pooled female and male flies was extracted using a high salt extraction protocol (Miller et al. 1988 (link)) and sheared with a Covaris S2 device (Covaris, Inc. Woburn, MA, USA). Libraries were prepared using the TruSeq DNA PCR-Free protocol (Illumina, San Diego, CA) and sequencing was performed with the Illumina HiSeq X Ten platform (Illumina, San Diego, CA). For an overview of the sequencing data used in this work see
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