.bam alignment files of Illumina and ONT sequencing of pREF plasmid

<p>The expression of genes encompasses their transcription into mRNA followed by translation into protein. In recent years, next-generation sequencing and mass spectrometry methods have profiled DNA, RNA and protein abundance in cells. However, there are currently no reference standards that are compatible across these genomic, transcriptomic and proteomic methods, and provide an integrated measure of gene expression. Here, we use synthetic biology principles to engineer a multi-omics control, termed <em>pREF</em>, that can act as a universal molecular standard for next-generation sequencing and mass spectrometry methods. The <em>pREF</em> sequence encodes 21 synthetic genes that can be <em>in vitro</em> transcribed into spike-in mRNA controls, and <em>in vitro</em> translated to generate matched protein controls. The synthetic genes provide qualitative controls that can measure sensitivity and quantitative accuracy of DNA, RNA and peptide detection. We demonstrate the use of <em>pREF</em> in metagenome DNA sequencing and RNA sequencing experiments and evaluate the quantification of proteins using mass spectrometry. Unlike previous spike-in controls, <em>pREF</em> can be independently propagated and the synthetic mRNA and protein controls can be sustainably prepared by recipient laboratories using common molecular biology techniques. Together, this provides the first universal synthetic standard able to integrate genomic, transcriptomic and proteomic methods.</p>