Intra‐genomic variation in G + C content and its implications for DNA stable isotope probing
2014
Youngblut, Nicholas D. | Buckley, Daniel H.
Combining deoxyribonucleic acid (DNA‐based) stable isotope probing (DNA‐SIP) with high‐throughput sequencing provides a powerful culture‐independent means to link microbial metabolic function to genomic information and taxonomic identity. DNA buoyant density (BD) in isopycnic gradients is dependent on both isotope incorporation and G + C content. G + C content varies across a genome but is constrained at rrn operons; hence, the ability to resolve isotopically labelled DNA from unlabelled DNA in SIP may vary between small subunit‐ribosomal nucleic acid (SSU rRNA) amplicon and shotgun‐read sequencing applications. We tested this hypothesis by evaluating the G + C content of genomic DNA fragments that encompassed either an SSU rRNA template (‘amplicon‐fragments’) or a shotgun read template (‘shotgun‐fragments’). We find that, contrary to expectations, the BD distribution of amplicon‐fragments is non‐normal and can be highly skewed. Furthermore, the BD distribution of amplicon‐fragments can differ substantially from that of shotgun‐fragments from the same genome. Our findings demonstrate the impact of G + C content on the downstream applications of DNA‐SIP, which will aid in proper experimental design and the development of statistical tests to accurately identify sequences derived from isotopically labelled DNA.
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