Genomic GC Research Articles

Four Inducible Promoters for Controlled Gene Expression in the Oleaginous Yeast Rhodotorula toruloides.

Rhodotorula (Rhodosporidium) toruloides is an oleaginous yeast with great biotechnological potential, capable of accumulating lipid up to 70% of its dry biomass, and of carotenoid biosynthesis. However, few molecular genetic tools are available for manipulation of this basidiomycete yeast and its high genomic GC content can make routine cloning difficult. We have developed plasmid vectors for transformation of R. toruloides which include elements for Saccharomyces cerevisiae in-yeast assembly; this method is robust to the assembly of GC-rich DNA and of large plasmids. Using such vectors we screened for controllable promoters, and identified inducible promoters from the genes NAR1, ICL1, CTR3, and MET16. These four promoters have independent induction/repression conditions and exhibit different levels and rates of induction in R. toruloides, making them appropriate for controllable transgene expression in different experimental situations. Nested deletions were used to identify regulatory regions in the four promoters, and to delimit the minimal inducible promoters, which are as small as 200 bp for the NAR1 promoter. The NAR1 promoter shows very tight regulation under repressed conditions as determined both by an EGFP reporter gene and by conditional rescue of a leu2 mutant. These new tools facilitate molecular genetic manipulation and controllable gene expression in R. toruloides.

Genome-wide comparative analysis of codon usage bias and codon context patterns among cyanobacterial genomes

With the increasing accumulation of genomic sequence information of prokaryotes, the study of codon usage bias has gained renewed attention. The purpose of this study was to examine codon selection pattern within and across cyanobacterial species belonging to diverse taxonomic orders and habitats. We performed detailed comparative analysis of cyanobacterial genomes with respect to codon bias. Our analysis reflects that in cyanobacterial genomes, A- and/or T-ending codons were used predominantly in the genes whereas G- and/or C-ending codons were largely avoided. Variation in the codon context usage of cyanobacterial genes corresponded to the clustering of cyanobacteria as per their GC content. Analysis of codon adaptation index (CAI) and synonymous codon usage order (SCUO) revealed that majority of genes are associated with low codon bias. Codon selection pattern in cyanobacterial genomes reflected compositional constraints as major influencing factor. It is also identified that although, mutational constraint may play some role in affecting codon usage bias in cyanobacteria, compositional constraint in terms of genomic GC composition coupled with environmental factors affected codon selection pattern in cyanobacterial genomes.

Lifestyle and DNA base composition in polychaetes.

A comparative analysis of polychaete species, classified as motile and low-motile forms, highlighted that the former were characterized not only by a higher metabolic rate (MR), but also by a higher genomic GC content. The fluctuation of both variables was not affected by the phylogenetic relationship of the species. Thus, present results further support that a very active lifestyle affects MR and GC at the same time, showing an unexpected similarity between invertebrates and vertebrates. In teleosts, indeed, a similar pattern has been also observed in comparisons of migratory and nonmigratory species. A cause-effect link between MR and GC has not yet been proved, but the fact that the two variables are significantly linked in all the organisms so far analyzed is, most probably, of relevant biological and evolutionary meaning. The present results fit very well within the frame of the metabolic rate hypothesis proposed to explain the DNA base composition variability among organisms. On the contrary, the thermostability hypothesis was not supported. At present, no data about the recombination rate in polychaetes were available to test the biased gene conversion (BGC hypothesis).

Gene Evolutionary Trajectories and GC Patterns Driven by Recombination in Zea mays.

Recombination occurring during meiosis is critical for creating genetic variation and plays an essential role in plant evolution. In addition to creating novel gene combinations, recombination can affect genome structure through altering GC patterns. In maize (Zea mays) and other grasses, another intriguing GC pattern exists. Maize genes show a bimodal GC content distribution that has been attributed to nucleotide bias in the third, or wobble, position of the codon. Recombination may be an underlying driving force given that recombination sites are often associated with high GC content. Here we explore the relationship between recombination and genomic GC patterns by comparing GC gene content at each of the three codon positions (GC1, GC2, and GC3, collectively termed GCx) to instances of a variable GC-rich motif that underlies double strand break (DSB) hotspots and to meiocyte-specific gene expression. Surprisingly, GCx bimodality in maize cannot be fully explained by the codon wobble hypothesis. High GCx genes show a strong overlap with the DSB hotspot motif, possibly providing a mechanism for the high evolutionary rates seen in these genes. On the other hand, genes that are turned on in meiosis (early prophase I) are biased against both high GCx genes and genes with the DSB hotspot motif, possibly allowing important meiotic genes to avoid DSBs. Our data suggests a strong link between the GC-rich motif underlying DSB hotspots and high GCx genes.

Switches in Genomic GC Content Drive Shifts of Optimal Codons under Sustained Selection on Synonymous Sites.

The major codon preference model suggests that codons read by tRNAs in high concentrations are preferentially utilized in highly expressed genes. However, the identity of the optimal codons differs between species although the forces driving such changes are poorly understood. We suggest that these questions can be tackled by placing codon usage studies in a phylogenetic framework and that bacterial genomes with extreme nucleotide composition biases provide informative model systems. Switches in the background substitution biases from GC to AT have occurred in Gardnerella vaginalis (GC = 32%), and from AT to GC in Lactobacillus delbrueckii (GC = 62%) and Lactobacillus fermentum (GC = 63%). We show that despite the large effects on codon usage patterns by these switches, all three species evolve under selection on synonymous sites. In G. vaginalis, the dramatic codon frequency changes coincide with shifts of optimal codons. In contrast, the optimal codons have not shifted in the two Lactobacillus genomes despite an increased fraction of GC-ending codons. We suggest that all three species are in different phases of an on-going shift of optimal codons, and attribute the difference to a stronger background substitution bias and/or longer time since the switch in G. vaginalis. We show that comparative and correlative methods for optimal codon identification yield conflicting results for genomes in flux and discuss possible reasons for the mispredictions. We conclude that switches in the direction of the background substitution biases can drive major shifts in codon preference patterns even under sustained selection on synonymous codon sites.

The Complete Chloroplast Genome Sequences of the Medicinal Plant Pogostemon cablin.

Pogostemon cablin, the natural source of patchouli alcohol, is an important herb in the Lamiaceae family. Here, we present the entire chloroplast genome of P. cablin. This genome, with 38.24% GC content, is 152,460 bp in length. The genome presents a typical quadripartite structure with two inverted repeats (each 25,417 bp in length), separated by one small and one large single-copy region (17,652 and 83,974 bp in length, respectively). The chloroplast genome encodes 127 genes, of which 107 genes are single-copy, including 79 protein-coding genes, four rRNA genes, and 24 tRNA genes. The genome structure, GC content, and codon usage of this chloroplast genome are similar to those of other species in the family, except that it encodes less protein-coding genes and tRNA genes. Phylogenetic analysis reveals that P. cablin diverged from the Scutellarioideae clade about 29.45 million years ago (Mya). Furthermore, most of the simple sequence repeats (SSRs) are short polyadenine or polythymine repeats that contribute to high AT content in the chloroplast genome. Complete sequences and annotation of P. cablin chloroplast genome will facilitate phylogenic, population and genetic engineering research investigations involving this particular species.

Cytochrome P450 genes in coronary artery diseases: Codon usage analysis reveals genomic GC adaptation

Establishing codon usage biases are imperative for understanding the etiology of coronary artery diseases (CAD) as well as the genetic factors associated with these diseases. The aim of this study was to evaluate the contribution of 18 responsible cytochrome P450 (CYP) genes for the risk of CAD. Effective number of codon (Nc) showed a negative correlation with both GC3 and synonymous codon usage order (SCUO) suggesting an antagonistic relationship between codon usage and Nc of genes. The dinucleotide analysis revealed that CG and TA dinucleotides have the lowest odds ratio in these genes. Principal component analysis showed that GC composition has a profound effect in separating the genes along the first major axis. Our findings revealed that mutational pressure and natural selection could possibly be the major factors responsible for codon bias in these genes. The study not only offers an insight into the mechanisms of genomic GC adaptation, but also illustrates the complexity of CYP genes in CAD.

Complete chloroplast genome sequence of a major economic species, Ziziphus jujuba (Rhamnaceae).

Ziziphus jujuba is an important woody plant with high economic and medicinal value. Here, we analyzed and characterized the complete chloroplast (cp) genome of Z. jujuba, the first member of the Rhamnaceae family for which the chloroplast genome sequence has been reported. We also built a web browser for navigating the cp genome of Z. jujuba ( http://bio.njfu.edu.cn/gb2/gbrowse/Ziziphus_jujuba_cp/ ). Sequence analysis showed that this cp genome is 161,466bp long and has a typical quadripartite structure of large (LSC, 89,120bp) and small (SSC, 19,348bp) single-copy regions separated by a pair of inverted repeats (IRs, 26,499bp). The sequence contained 112 unique genes, including 78 protein-coding genes, 30 transfer RNAs, and four ribosomal RNAs. The genome structure, gene order, GC content, and codon usage are similar to other typical angiosperm cp genomes. A total of 38 tandem repeats, two forward repeats, and three palindromic repeats were detected in the Z. jujuba cp genome. Simple sequence repeat (SSR) analysis revealed that most SSRs were AT-rich. The homopolymer regions in the cp genome of Z. jujuba were verified and manually corrected by Sanger sequencing. One-third of mononucleotide repeats were found to be erroneously sequenced by the 454 pyrosequencing, which resulted in sequences of 1-4 bases shorter than that by the Sanger sequencing. Analyzing the cp genome of Z. jujuba revealed that the IR contraction and expansion events resulted in ycf1 and rps19 pseudogenes. A phylogenetic analysis based on 64 protein-coding genes showed that Z. jujuba was closely related to members of the Elaeagnaceae family, which will be helpful for phylogenetic studies of other Rosales species. The complete cp genome sequence of Z. jujuba will facilitate population, phylogenetic, and cp genetic engineering studies of this economic plant.

Optimized Illumina PCR-free library preparation for bacterial whole genome sequencing and analysis of factors influencing de novo assembly

BackgroundNext-generation sequencing (NGS) technology has paved the way for rapid and cost-efficient de novo sequencing of bacterial genomes. In particular, the introduction of PCR-free library preparation procedures (LPPs) lead to major improvements as PCR bias is largely reduced. However, in order to facilitate the assembly of Illumina paired-end sequence data and to enhance assembly performance, an increase of insert sizes to facilitate the repeat bridging and resolution capabilities of current state of the art assembly tools is needed. In addition, information concerning the relationships between genomic GC content, library insert size and sequencing quality as well as the influence of library insert size, read length and sequencing depth on assembly performance would be helpful to specifically target sequencing projects.ResultsOptimized DNA fragmentation settings and fine-tuned resuspension buffer to bead buffer ratios during fragment size selection were integrated in the Illumina TruSeq® DNA PCR-free LPP in order to produce sequencing libraries varying in average insert size for bacterial genomes within a range of 35.4–73.0 % GC content. The modified protocol consumes only half of the reagents per sample, thus doubling the number of preparations possible with a kit. Examination of different libraries revealed that sequencing quality decreases with increased genomic GC content and with larger insert sizes. The estimation of assembly performance using assembly metrics like corrected NG50 and NGA50 showed that libraries with larger insert sizes can result in substantial assembly improvements as long as appropriate assembly tools are chosen. However, such improvements seem to be limited to genomes with a low to medium GC content. A positive trend between read length and assembly performance was observed while sequencing depth is less important, provided a minimum coverage is reached.ConclusionsBased on the optimized protocol developed, sequencing libraries with flexible insert sizes and lower reagent costs can be generated. Furthermore, increased knowledge about the interplay of sequencing quality, insert size, genomic GC content, read length, sequencing depth and the assembler used will help molecular biologists to set up an optimal experimental and analytical framework with respect to Illumina next-generation sequencing of bacterial genomes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-016-2072-9) contains supplementary material, which is available to authorized users.

The Frequency of Internal Shine-Dalgarno-like Motifs in Prokaryotes.

In prokaryotes, translation initiation typically depends on complementary binding between a G-rich Shine–Dalgarno (SD) motif in the 5′ untranslated region of mRNAs, and the 3′ tail of the 16S ribosomal RNA (the anti-SD sequence). In some cases, internal SD-like motifs in the coding region generate “programmed” ribosomal pauses that are beneficial for protein folding or accurate targeting. On the other hand, such pauses can also reduce protein production, generating purifying selection against internal SD-like motifs. This selection should be stronger in GC-rich genomes that are more likely to harbor the G-rich SD motif. However, the nature and consequences of selection acting on internal SD-like motifs within genomes and across species remains unclear. We analyzed the frequency of SD-like hexamers in the coding regions of 284 prokaryotes (277 with known anti-SD sequences and 7 without a typical SD mechanism). After accounting for GC content, we found that internal SD-like hexamers are avoided in 230 species, including three without a typical SD mechanism. The degree of avoidance was higher in GC-rich genomes, mesophiles, and N-terminal regions of genes. In contrast, 54 species either showed no signature of avoidance or were enriched in internal SD-like motifs. C-terminal gene regions were relatively enriched in SD-like hexamers, particularly for genes in operons or those followed closely by downstream genes. Together, our results suggest that the frequency of internal SD-like hexamers is governed by multiple factors including GC content and genome organization, and further empirical work is necessary to understand the evolution and functional roles of these motifs.

A Recent Whole-Genome Duplication Divides Populations of a Globally Distributed Microsporidian.

The Microsporidia are a major group of intracellular fungi and important parasites of animals including insects, fish, and immunocompromised humans. Microsporidian genomes have undergone extreme reductive evolution but there are major differences in genome size and structure within the group: some are prokaryote-like in size and organisation (<3 Mb of gene-dense sequence) while others have more typically eukaryotic genome architectures. To gain fine-scale, population-level insight into the evolutionary dynamics of these tiny eukaryotic genomes, we performed the broadest microsporidian population genomic study to date, sequencing geographically isolated strains of Spraguea, a marine microsporidian infecting goosefish worldwide. Our analysis revealed that population structure across the Atlantic Ocean is associated with a conserved difference in ploidy, with American and Canadian isolates sharing an ancestral whole genome duplication that was followed by widespread pseudogenisation and sorting-out of paralogue pairs. While past analyses have suggested de novo gene formation of microsporidian-specific genes, we found evidence for the origin of new genes from noncoding sequence since the divergence of these populations. Some of these genes experience selective constraint, suggesting the evolution of new functions and local host adaptation. Combining our data with published microsporidian genomes, we show that nucleotide composition across the phylum is shaped by a mutational bias favoring A and T nucleotides, which is opposed by an evolutionary force favoring an increase in genomic GC content. This study reveals ongoing dramatic reorganization of genome structure and the evolution of new gene functions in modern microsporidians despite extensive genomic streamlining in their common ancestor.

The mutation spectrum in genomic late replication domains shapes mammalian GC content.

Genome sequence compositions and epigenetic organizations are correlated extensively across multiple length scales. Replication dynamics, in particular, is highly correlated with GC content. We combine genome-wide time of replication (ToR) data, topological domains maps and detailed functional epigenetic annotations to study the correlations between replication timing and GC content at multiple scales. We find that the decrease in genomic GC content at large scale late replicating regions can be explained by mutation bias favoring A/T nucleotide, without selection or biased gene conversion. Quantification of the free dNTP pool during the cell cycle is consistent with a mechanism involving replication-coupled mutation spectrum that favors AT nucleotides at late S-phase. We suggest that mammalian GC content composition is shaped by independent forces, globally modulating mutation bias and locally selecting on functional element. Deconvoluting these forces and analyzing them on their native scales is important for proper characterization of complex genomic correlations.

On the genome base composition of teleosts: the effect of environment and lifestyle.

BackgroundThe DNA base composition is well known to be highly variable among organisms. Bio-physic studies on the effect of the GC increments on the DNA structure have shown that GC-richer DNA sequences are more bendable. The result was the keystone of the hypothesis proposing the metabolic rate as the major force driving the GC content variability, since an increased resistance to the torsion stress is mainly required during the transcription process to avoid DNA breakage. Hence, the aim of the present work is to test if both salinity and migration, suggested to affect the metabolic rate of teleostean fishes, affect the average genomic GC content as well. Moreover, since the gill surface has been reported to be a major morphological expression of metabolic rate, this parameter was also analyzed in the light of the above hypothesis.ResultsTeleosts living in different environments (freshwater and seawater) and with different lifestyles (migratory and non-migratory) were analyzed studying three variables: routine metabolic rate, gill area and genomic GC-content, none of them showing a phylogenetic signal among fish species. Routine metabolic rate, specific gill area and average genomic GC were higher in seawater than freshwater species. The same trend was observed comparing migratory versus non-migratory species. Crossing salinity and lifestyle, the active migratory species living in seawater show coincidentally the highest routine metabolic rate, the highest specific gill area and the highest average genomic GC content.ConclusionsThe results clearly highlight that environmental factors (salinity) and lifestyle (migration) affect not only the physiology (i.e. the routine metabolic rate), and the morphology (i.e. gill area) of teleosts, but also basic genome feature (i.e. the GC content), thus opening to an interesting liaison among the three variables in the light of the metabolic rate hypothesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2537-1) contains supplementary material, which is available to authorized users.

Complete chloroplast genome sequences of Eucommia ulmoides: genome structure and evolution

Eucommia ulmoides is an important traditional medicinal plant that is used for the production of locative Eucommia rubber. In this study, the complete chloroplast (cp) genome sequence of E. ulmoides was obtained by total DNA sequencing; this is the first cp genome sequence of the order Garryales. The cp genome of E. ulmoides was 163,341 bp long and included a pair of inverted repeat (IR) regions (31,300 bp), one large single copy (LSC) region (86,592 bp), and one small single copy (SSC) region (14,149 bp). The genome structure and GC content were similar to those of typical angiosperm cp genomes and contained 115 unique genes, including 80 protein-coding genes, 31 transfer RNA (tRNAs), and four ribosomal RNA (rRNAs). Compared with the entire cp genome sequence, three unique genome rearrangements were observed in the LSC region. Moreover, compared with the Sesamum and Nicotiana cp genomes, E. ulmoides contained no indels in the IR regions, and variable regions were identified in noncoding regions. The E. ulmoides cp genome showed extreme expansion at the IR/SSC boundary owing to the integration of an additional complete gene, ycf1. Twenty-nine simple sequence repeats (SSRs) were identified in the E. ulmoides cp genome. In addition, 36 protein-coding genes were used for phylogenetic inference, supporting a sister relationship between E. ulmoides and Aucuba, which belongs to Euasterids I. In summary, we described the complete cp genome sequence of E. ulmoides; this information will be useful for phylogenetic and evolutionary studies.

In silico genome-wide identification and analysis of microsatelliterepeats in the largest RNA virus family (Closteroviridae)

Microsatellites are known to exhibit ubiquitous presence across all kingdoms of life, including viruses. Members of the family Closteroviridae are the largest RNA viruses and severely affect different agricultural crops worldwide, including1397645907citrus, grapevine, and vegetables. Here we identified and systematically analyzed the nature and distribution of both simple and complex microsatellites present in the complete genome of 36 species belonging to Closteroviridae. Our results showed, in all analyzed genomes, that neither genome size nor GC content had any influence on number, relative abundance, or relative density of microsatellites. For each genome, dinucleotide repeats were found to be highly predominant and AT/TA and AG/GA were the two most abundant dinucleotide repeat motifs. Repeats larger than trinucleotide were relatively rare in these viral genomes. Comparative study of occurrence, abundance, and density of microsatellites among available RNA and DNA viral genomes indicated that simple repeats were less abundant in genomes of Closteroviridae. To our knowledge, this is the first analysis of microsatellites occurring in the largest viral genome that infects plants. Characterization of such variations in repeat sequences would be important in deciphering the origin, mutational processes, and role of repeat sequences in viral genomes.

The production of S-equol from daidzein is associated with a cluster of three genes in Eggerthella sp. YY7918.

Daidzein (DZN) is converted to equol (EQL) by intestinal bacteria. We previously reported that Eggerthella sp. YY7918, which is found in human feces, is an EQL-producing bacterium and analyzed its whole genomic sequence. We found three coding sequences (CDSs) in this bacterium that showed 99% similarity to the EQL-producing enzymes of Lactococcus sp. 20-92. These identified CDSs were designated eqlA, eqlB, and eqlC and thought to encode daidzein reductase (DZNR), dihydrodaidzein reductase (DHDR), and tetrahydrodaidzein reductase (THDR), respectively. These genes were cloned into pColdII. Recombinant plasmids were then introduced into Escherichia coli BL21 (DE3) and DZNR, DHDR, and THDR were expressed and purified by 6×His-Tag chromatography. We confirmed that these three enzymes were involved in the conversion of DZN to EQL. Purified DZNR converted DZN to dihydrodaizein (DHD) in the presence of NADPH. DHDR converted DHD to tetrahydrodaizein (THD) in the presence of NADPH. Neither enzyme showed activities with NADH. THDR converted THD in the absence of cofactors, NAD(P)H, and also produced DHD as a by-product. Thus, we propose that THDR is not a reductase but a new type of dismutase. The GC content of these clusters was 64%, similar to the overall genomic GC content for Eggerthella and Coriobacteriaceae (56–60%), and higher than that for Lactococcus garvieae (39%), even though the gene cluster showed 99% similarity to that in Lactococcus sp. 20-92. Taken together, our results indicate that the gene cluster associated with EQL production evolved in high-GC bacteria including Coriobacteriaceae and was then laterally transferred to Lactococcus sp. 20-92.

Genome-wide maps of nucleosomes of the trichostatin A treated and untreated archiascomycetous yeast &lt;em&gt;Saitoella complicata&lt;/em&gt;

We investigated the effects of trichostatin A (TSA) on gene expression and nucleosome position in the archiascomycetous yeast Saitoella complicata. The expression levels of 154 genes increased in a TSA-concentration-dependent manner, while the levels of 131 genes decreased. Conserved genes between S. complicata and Schizosaccharomyces pombe were more commonly TSA-concentration-dependent downregulated genes than upregulated genes. We calculated the correlation coefficients of nucleosome position profiles within 300 nucleotides (nt) upstream of a translational start of S. complicata grown in the absence and the presence of TSA (3 μg/mL). We found that 20 (13.0%) of the 154 TSA-concentration-dependent upregulated genes and 22 (16.8%) of the 131 downregulated genes had different profiles (r 0.8). We did not observe a GC content bias between the 300 nt upstream of the translational start of the TSA-concentration-dependent genes with conserved nucleosome positioning and the genes with different nucleosome positioning, suggesting that TSA-induced nucleosome position change is likely not related to DNA sequence. Most gene promoters maintained their nucleosome positioning even after TSA treatment, which may be related to DNA sequence. Enriched and depleted dinucleotides distribution of S. complicata around the midpoints of highly positioned nucleosome dyads was not similar to that of the phylogenetically close yeast Schizosaccharomyces pombe but similar to the basidiomycete Mixia osmundae, which has similar genomic GC content to that of S. complicata.

Use of MinION data to assemble the 67% GC genome sequence of Bordetella pertussis

Bordetella pertussi s, the major casuative agent of whooping cough, contains about 280 1kb repeats. These confound genome assembly using Illumina data alone. We hope to get round this problem using long reads from Oxford Nanopore's MinION device.

Composition of distinct sub-proteomes in Myxococcus xanthus: metabolic cost and amino acid availability.

Subsets of proteins involved in distinct functional processes are subject to different selective pressures. We investigated whether there is an amino acid composition bias (AACB) inherent in discrete subsets of proteins, and whether we could identify changing patterns of AACB during the life cycle of the social bacterium Myxococcus xanthus. We quantitatively characterised the cellular, soluble secreted, and outer membrane vesicle (OMV) sub-proteomes of M. xanthus, identifying 315 proteins. The AACB of the cellular proteome differed only slightly from that deduced from the genome, suggesting that genome-inferred proteomes can accurately reflect the AACB of their host. Inferred AA deficiencies arising from prey consumption were exacerbated by the requirements of the 68%GC genome, whose character thus seems to be selected for directly rather than via the proteome. In our analysis, distinct subsets of the proteome (whether segregated spatially or temporally) exhibited distinct AACB, presumably tailored according to the needs of the organism's lifestyle and nutrient availability. Secreted AAs tend to be of lower cost than those retained in the cell, except for the early developmental A-signal, which is a particularly costly sub-proteome. We propose a model of AA reallocation during the M. xanthus life cycle, involving ribophagy during early starvation and sequestration of limiting AAs within cells during development.

New perspectives on microbial community distortion after whole-genome amplification.

Whole-genome amplification (WGA) has become an important tool to explore the genomic information of microorganisms in an environmental sample with limited biomass, however potential selective biases during the amplification processes are poorly understood. Here, we describe the effects of WGA on 31 different microbial communities from five biotopes that also included low-biomass samples from drinking water and groundwater. Our findings provide evidence that microbiome segregation by biotope was possible despite WGA treatment. Nevertheless, samples from different biotopes revealed different levels of distortion, with genomic GC content significantly correlated with WGA perturbation. Certain phylogenetic clades revealed a homogenous trend across various sample types, for instance Alpha- and Betaproteobacteria showed a decrease in their abundance after WGA treatment. On the other hand, Enterobacteriaceae, an important biomarker group for fecal contamination in groundwater and drinking water, were strongly affected by WGA treatment without a predictable pattern. These novel results describe the impact of WGA on low-biomass samples and may highlight issues to be aware of when designing future metagenomic studies that necessitate preceding WGA treatment.