Combined analysis of microbial metagenomic and metatranscriptomic sequencing data to assess in situ physiological conditions in the premature infant gut

Abstract

Microbes alter their transcriptomic profiles in response to the environment. The physiological conditions experienced by a microbial community can thus be inferred using meta-transcriptomic sequencing by comparing transcription levels of specifically chosen genes. However, this analysis requires accurate reference genomes to identify the specific genes from which RNA reads originate. In addition, such an analysis should avoid biases in transcript counts related to differences in organism abundance. In this study we describe an approach to address these difficulties. Sample-specific meta-genomic assembled genomes (MAGs) were used as reference genomes to accurately identify the origin of RNA reads, and transcript ratios of genes with opposite transcription responses were compared to eliminate biases related to differences in organismal abundance, an approach hereafter named the "diametric ratio" method. We used this approach to probe the environmental conditions experienced by Escherichia spp. in the gut of 4 premature infants, 2 of whom developed necrotizing enterocolitis (NEC), a severe inflammatory intestinal disease. We analyzed twenty fecal samples taken from four premature infants (4-6 time points from each infant), and found significantly higher diametric ratios of genes associated with low oxygen levels in samples of infants later diagnosed with NEC than in samples without NEC. We also show this method can be used for examining other physiological conditions, such as exposure to nitric oxide and osmotic pressure. These study results should be treated with caution, due to the presence of confounding factors that might also distinguish between NEC and control infants. Nevertheless, together with benchmarking analyses, we show here that the diametric ratio approach can be applied for evaluating the physiological conditions experienced by microbes in situ. Results from similar studies can be further applied for designing diagnostic methods to detect NEC in its early developmental stages.

Fig 1. Transcriptional response to oxygen by Escherichia spp. in the gut of NEC and control premature infants.

(A) Diametric ratios were compared between NEC and control infants in each time block (short lines above) and across all time points (longer lines). Distributions of diametric ratios were compared using Welch’s t-test with Bonferroni correction. Asterisks and double asterisks (*, **) represent p < 0.05 and p <0.01, respectively. (B) Diametric ratios of cydAB and cyoABCD transcript abundances of RNA reads mapped to gene sequences of Escherichia spp. genomes found in infants’ gut. Filtering of reads with more than 1 miss matches was applied. (C) Diametric ratios of cydAB and cyoABCD transcript abundances of RNA reads mapped to gene sequences of E. coli (K-12 MG1655) downloaded from KEGG (Kyoto Encyclopedia of Genes and Genomes) database. Filtering of reads with more than 1 miss matches was applied. (D) Diametric ratios of cydAB and cyoABCD transcript abundances of RNA reads mapped to gene sequences of Escherichia spp. genomes found in infants’ gut. No filtering of reads with miss matches was applied.

Fig 2. Transcriptional response to oxygen, Nitric oxide and osmotic conditions by Escherichia spp. in the gut of NEC and control premature infants.

(A) Diametric ratios were compared between NEC and control infants in each time block (short lines above) and across all time points (longer lines). Distributions of diametric ratios were compared using Welch’s t-test with Bonferroni correction. Asterisks and double asterisks (*, **) represent p < 0.05 and p <0.01, respectively.(A) Diametric ratios of arcA and fnr transcript abundances of RNA reads mapped to sequences of Escherichia spp. genomes found in infants’ gut. (B) Diametric ratios of nrdDG and nrdAB transcript abundances of RNA reads mapped to sequences of Escherichia spp. genomes found in infants’ gut. (C) Diametric ratios of ompC and ompF transcript abundances of RNA reads mapped to gene sequences of Escherichia spp. genomes found in infants’ gut. (D) Diametric ratios of norVW and norV transcript abundances of RNA reads mapped to gene sequences of Escherichia spp. genomes found in infants’ gut.