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Is amplification bias consequential in transposon sequencing (TnSeq) assays? A case study with a Staphylococcus aureus TnSeq library subjected to PCR-based and amplification-free enrichment methods.

ABSTRACT: As transposon sequencing (TnSeq) assays have become prolific in the microbiology field, it is of interest to scrutinize their potential drawbacks. TnSeq data consist of millions of nucleotide sequence reads that are generated by PCR amplification of transposon-genomic junctions. Reads mapping to the junctions are enumerated thus providing information on the number of transposon insertion mutations in each individual gene. Here we explore the possibility that PCR amplification of transposon insertions in a TnSeq library skews the results by introducing bias into the detection and/or enumeration of insertions. We compared the detection and frequency of mapped insertions when altering the number of PCR cycles, and when including a nested PCR, in the enrichment step. Additionally, we present nCATRAs - a novel, amplification-free TnSeq method where the insertions are enriched via CRISPR/Cas9-targeted transposon cleavage and subsequent Oxford Nanopore MinION sequencing. nCATRAs achieved 54 and 23% enrichment of the transposons and transposon-genomic junctions, respectively, over background genomic DNA. These PCR-based and PCR-free experiments demonstrate that, overall, PCR amplification does not significantly bias the results of TnSeq insofar as insertions in the majority of genes represented in our library were similarly detected regardless of PCR cycle number and whether or not PCR amplification was employed. However, the detection of a small subset of genes which had been previously described as essential is sensitive to the number of PCR cycles. We conclude that PCR-based enrichment of transposon insertions in a TnSeq assay is reliable, but researchers interested in profiling putative essential genes should carefully weigh the number of amplification cycles employed in their library preparation protocols. In addition, nCATRAs is comparable to traditional PCR-based methods (Kendall's correlation=0.896-0.897) although the latter remain superior owing to their accessibility and high sequencing depth.

SUBMITTER: Alkam D

PROVIDER: S-EPMC8627206 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:TnSeq is a widely used methodology for determining gene essentiality, conditional fitness, and genetic interactions in bacteria. The Himar1 transposon is restricted to insertions at TA dinucleotides, but otherwise, few site-specific biases have been identified. As a result, most analytical approaches assume that insertions are expected to be randomly distributed among TA sites in nonessential regions. However, through analysis of Himar1 transposon libraries in Mycobacterium tuberculosis, we demonstrate that there are site-specific biases that affect the frequency of insertion of the Himar1 transposon at different TA sites. We use machine learning and statistical models to characterize patterns in the nucleotides surrounding TA sites that correlate with high or low insertion counts. We then develop a quantitative model based on these patterns that can be used to predict the expected counts at each TA site based on nucleotide context, which can explain up to half of the variance in insertion counts. We show that these insertion preferences exist in Himar1 TnSeq data sets from other mycobacterial and nonmycobacterial species. We present an improved method for identification of essential genes, called TTN-Fitness, that can better distinguish true biological fitness effects by comparing observed counts to expected counts based on our site-specific model of insertion preferences. Compared to previous essentiality methods, TTN-Fitness can make finer distinctions among genes whose disruption causes a fitness defect (or advantage), separating them out from the large pool of nonessentials, and is able to classify many smaller genes (with few TA sites) that were previously characterized as uncertain. IMPORTANCE When using the Himar1 transposon to create transposon insertion mutant libraries, it is known that the transposon is restricted to insertions at TA dinucleotide sites throughout the genome, and the absence of insertions is used to infer which genes are essential (or conditionally essential) in a bacterial organism. It is widely assumed that insertions in nonessential regions are otherwise random, and this assumption is used as the basis of several methods for statistical analysis of TnSeq data. In this paper, we show that the nucleotide sequence surrounding TA sites influences the magnitude of insertions, and these Himar1 insertion preferences (sequence biases) can partially explain why some sites have higher counts than others. We use this predictive model to make improved estimates of the fitness effects of genes, which help make finer distinctions of the phenotype and biological consequences of disruption of nonessential genes.

Project description:Simple Summary Inhalation of particulate matter (PM2.5) is known to cause cardiac effects and exacerbate any pre-existing cardiac diseases. However, the severity of toxicity depends on how much PM2.5 has reached the blood circulation and then finally the heart. The consequential impact of PM2.5 on the heart depends on its potential to alter cardiac oxidative stress and mitochondrial function that in turn controls the contractile function of the heart. In fact, this effect is partly concentration dependent. In the present study, we demonstrated that blood borne PM2.5 can inflict cardiac injury and compromise physiological response. Moreover, the results from our study show that cardiac tolerance to resist ischemia reperfusion injury (IR) is low in PM2.5 administered rat hearts. Cellular level analysis of the data suggests that PM2.5 gets deposited in the mitochondria which in turn increases the oxidative stress by disturbing redox couple and inducing mitochondrial dysfunction. In fact, the higher pathological changes occur with the direct entry of PM into the myocardium. Abstract A previous study has reported that exposure to PM2.5 from diesel exhaust (diesel particulate matter (DPM)) for 21 days can deteriorate the cardiac recovery from myocardial ischemia reperfusion injury (IR), where the latter is facilitated by the efficiency of mitochondrial subpopulations. Many investigators have demonstrated that IR impact on cardiac mitochondrial subpopulations is distinct. In the present study, we decipher the role of PM2.5 on IR associated mitochondrial dysfunction at the subpopulation level by administrating PM2.5 directly to isolated female rat hearts via KH buffer. Our results demonstrated that PM2.5 administered heart (PM_C) severely deteriorated ETC enzyme activity (NQR, SQR, QCR, and COX) and ATP level in both IFM and SSM from the normal control. Comparatively, the declined activity was prominent in IFM fraction. Moreover, in the presence of IR (PM_IR), mitochondrial oxidative stress was higher in both subpopulations from the normal, where the IFM fraction of mitochondria experienced elevated oxidative stress than SSM. Furthermore, we assessed the in vitro protein translation capacity of IFM and SSM and found a declined ability in both subpopulations where the inability of IFM was significant in both PM_C and PM_IR groups. In support of these results, the expression of mitochondrial genes involved in fission, fusion, and mitophagy events along with the DNA maintenance genes such as GUF1, LRPPRC, and HSD17-b10 were significantly altered from the control. Based on the above results, we conclude that PM2.5 administration to the heart inflicted mitochondrial damage especially to the IFM fraction, that not only deteriorated the cardiac physiology but also reduced its ability to resist IR injury.

Project description:BackgroundViruses are key players regulating microbial ecosystems. Exploration of viral assemblages is now possible thanks to the development of metagenomics, the most powerful tool available for studying viral ecology and discovering new viruses. Unfortunately, several sources of bias lead to the misrepresentation of certain viruses within metagenomics workflows, hindering the shift from merely descriptive studies towards quantitative comparisons of communities. Therefore, benchmark studies on virus enrichment and random amplification protocols are required to better understand the sources of bias.ResultsWe assessed the bias introduced by viral enrichment on mock assemblages composed of seven DNA viruses, and the bias from random amplification methods on human saliva DNA viromes, using qPCR and deep sequencing, respectively. While iodixanol cushions and 0.45 μm filtration preserved the original composition of nuclease-protected viral genomes, low-force centrifugation and 0.22 μm filtration removed large viruses. Comparison of unamplified and randomly amplified saliva viromes revealed that multiple displacement amplification (MDA) induced stochastic bias from picograms of DNA template. However, the type of bias shifted to systematic using 1 ng, with only a marginal influence by amplification time. Systematic bias consisted of over-amplification of small circular genomes, and under-amplification of those with extreme GC content, a negative bias that was shared with the PCR-based sequence-independent, single-primer amplification (SISPA) method. MDA based on random priming provided by a DNA primase activity slightly outperformed those based on random hexamers and SISPA, which may reflect differences in ability to handle sequences with extreme GC content. SISPA viromes showed uneven coverage profiles, with high coverage peaks in regions with low linguistic sequence complexity. Despite misrepresentation of certain viruses after random amplification, ordination plots based on dissimilarities among contig profiles showed perfect overlapping of related amplified and unamplified saliva viromes and strong separation from unrelated saliva viromes. This result suggests that random amplification bias has a minor impact on beta diversity studies.ConclusionsBenchmark analyses of mock and natural communities of viruses improve understanding and mitigate bias in metagenomics surveys. Bias induced by random amplification methods has only a minor impact on beta diversity studies of human saliva viromes.

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Is amplification bias consequential in transposon sequencing (TnSeq) assays? A case study with a Staphylococcus aureus TnSeq library subjected to PCR-based and amplification-free enrichment methods.

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