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BACKGROUND: We have been documenting the biological responses to low levels of radiation (natural background) and very low level radiation (below background), and thus these studies are testing mild external stimuli to which we would expect relatively mild biological responses. We recently published a transcriptome software comparison study based on RNA-Seqs from a below background radiation treatment of two model organisms, E. coli and C. elegans (Thawng and Smith, BMC Genomics 23:452, 2022). We reported DNAstar-D (Deseq2 in the DNAstar software pipeline) to be the more conservative, realistic tool for differential gene expression compared to other transcriptome software packages (CLC, Partek and DNAstar-E (using edgeR). Here we report two follow-up studies (one with a new model organism, Aedes aegypti and another software package (Azenta) on transcriptome responses from varying dose rates using three different sources of natural radiation. RESULTS: When E. coli was exposed to varying levels of K40, we again found that the DNAstar-D pipeline yielded a more conservative number of DEGs and a lower fold-difference than the CLC pipeline and DNAstar-E run in parallel. After a 30 read minimum cutoff criterion was applied to the data, the number of significant DEGs ranged from 0 to 81 with DNAstar-D, while the number of significant DEGs ranged from 4 to 117 and 14 to 139 using DNAstar-E and the CLC pipelines, respectively. In terms of the extent of expression, the highest foldchange DEG was observed in DNAstar-E with 19.7-fold followed by 12.5-fold in CLC and 4.3-fold in DNAstar-D. In a recently completed study with Ae. Aegypti and using another software package (Azenta), we analyzed the RNA-Seq response to similar sources of low-level radiation and again found the DNAstar-D pipeline to give the more conservative number and fold-expression of DEGs compared to other softwares. The number of significant DEGs ranged 31-221 in Azenta and 31 to 237 in CLC, 19-252 in DNAstar-E and 0-67 in DNAStar-D. The highest fold-change of DEGs were found in CLC (1,350.9-fold), with DNAstar-E (5.9 -fold) and Azenta (5.5-fold) intermediate, and the lowest levels of expression (4-fold) found in DNAstar-D. CONCLUSIONS: This study once again highlights the importance of choosing appropriate software for transcriptome analysis. Using three different biological models (bacteria, nematode and mosquito) in four different studies testing very low levels of radiation (Van Voorhies et al., Front Public Health 8:581796, 2020; Thawng and Smith, BMC Genomics 23:452, 2022; current study), the CLC software package resulted in what appears to be an exaggerated gene expression response in terms of numbers of DEGs and extent of expression. Setting a 30-read cutoff diminishes this exaggerated response in most of the software tested. We have further affirmed that DNAstar-Deseq2 gives a more conservative transcriptome expression pattern which appears more suitable for studies expecting subtle gene expression patterns.

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BACKGROUND: In this comparative study we evaluate the performance of four software tools: DNAstar-D (DESeq2), DNAstar-E (edgeR), CLC Genomics and Partek Flow for identification of differentially expressed genes (DEGs) using a transcriptome of E. coli. The RNA-seq data are from the effect of below-background radiation 5.5 nGy total dose (0.2nGy/hr) on E. coli grown shielded from natural radiation 655 m below ground in a pre-World War II steel vault. The gene expression response to three supplemented sources of radiation designed to mimic natural background, 1952 - 5720 nGy in total dose (71-208 nGy/hr), are compared to this "radiation-deprived" treatment. In addition, RNA-seq data of Caenorhabditis elegans nematode from similar radiation treatments was analyzed by three of the software packages. RESULTS: In E. coli, the four software programs identified one of the supplementary sources of radiation (KCl) to evoke about 5 times more transcribed genes than the minus-radiation treatment (69-114 differentially expressed genes, DEGs), and so the rest of the analyses used this KCl vs "Minus" comparison. After imposing a 30-read minimum cutoff, one of the DNAStar options shared two of the three steps (mapping, normalization, and statistic) with Partek Flow (they both used median of ratios to normalize and the DESeq2 statistical package), and these two programs identified the highest number of DEGs in common with each other (53). In contrast, when the programs used different approaches in each of the three steps, between 31 and 40 DEGs were found in common. Regarding the extent of expression differences, three of the four programs gave high fold-change results (15-178 fold), but one (DNAstar's DESeq2) resulted in more conservative fold-changes (1.5-3.5). In a parallel study comparing three qPCR commercial validation software programs, these programs also gave variable results as to which genes were significantly regulated. Similarly, the C. elegans analysis showed exaggerated fold-changes in CLC and DNAstar's edgeR while DNAstar-D was more conservative. CONCLUSIONS: Regarding the extent of expression (fold-change), and considering the subtlety of the very low level radiation treatments, in E. coli three of the four programs gave what we consider exaggerated fold-change results (15 - 178 fold), but one (DNAstar's DESeq2) gave more realistic fold-changes (1.5-3.5). When RT-qPCR validation comparisons to transcriptome results were carried out, they supported the more conservative DNAstar-D's expression results. When another model organism's (nematode) response to these radiation differences was similarly analyzed, DNAstar-D also resulted in the most conservative expression patterns. Therefore, we would propose DESeq2 ("DNAstar-D") as an appropriate software tool for differential gene expression studies for treatments expected to give subtle transcriptome responses.

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