Oxford Nanopore long reads of simulated bacterial communities from fresh spinach and surface water were generated (R9.4.1+SQK-LSK109 and R10.4+SQK-LSK112; 0.5, one, and two million reads). Salmonella enterica serotype Heidelberg, Montevideo, or Typhimurium was included alone or in combination in the spinach community, while the water community harbored Pseudomonas aeruginosa.

As the amount of metagenomic sequencing continues to increase, there is a growing need for tools that help biologists make sense of the data. Specifically, researchers are often interested in the potential of a microbial community to carry out a metabolic reaction, but this analysis requires knitting together multiple software tools into a complex pipeline. Thanos offers a user-friendly R package designed for the pathway-centric analysis and visualization of the functions encoded within metagenomic samples. It allows researchers to go beyond taxonomic profiles and find out, quantitatively, which pathways are prevalent in an environment, as well as comparing different environments in terms of their functional potential. The analysis is based on the sequencing depth of the genes of interest, either in the metagenome-assembled genomes (MAGs) or in the assembled reads (contigs), using a normalization strategy that enables comparison across samples. The package can import the data from multiple formats and offers functions for the visualization of the results as bar plots of the functional profile, box plots of compare functions across samples, and annotated pathway graphs. By streamlining the analysis of the functional potential encoded in microbial communities, Thanos can enable impactful discoveries in all the fields touched by metagenomics, from human health to the environmental sciences.

Paired-end short reads of Illumina HiSeq, MiSeq, and NovaSeq of simulated bacterial communities from fresh spinach and surface water were generated in silico at various sequencing depths. Multidrug-resistant Salmonella enterica serotype Indiana was included in the spinach community, while the water community contained multidrug-resistant Pseudomonas aeruginosa.

Shotgun metagenomics sequencing experiments are finding a wide range of applications. Nonetheless, there are still limited guidelines regarding the number of sequences needed to acquire meaningful information for taxonomic profiling and antimicrobial resistance gene (ARG) identification. In this study, we explored this issue in the context of oral microbiota by sequencing with a very high number of sequences (~ 100 million), four human plaque samples, and one microbial community standard and by evaluating the performance of microbial identification and ARGs detection through a downsampling procedure. When investigating the impact of a decreasing number of sequences on quantitative taxonomic profiling in the microbial community standard datasets, we found some discrepancies in the identified microbial species and their abundances when compared to the expected ones. Such differences were consistent throughout downsampling, suggesting their link to taxonomic profiling methods limitations. Overall, results showed that the number of sequences has a great impact on metagenomic samples at the qualitative (i.e., presence/absence) level in terms of loss of information, especially in experiments having less than 40 million reads, whereas abundance estimation was minimally affected, with only slight variations observed in low-abundance species. The presence of ARGs was also assessed: a total of 133 ARGs were identified. Notably, 23% of them inconsistently resulted as present or absent across downsampling datasets of the same sample. Moreover, over half of ARGs were lost in datasets having less than 20 million reads. This study highlights the importance of carefully considering sequencing aspects and suggests some guidelines for designing shotgun metagenomics experiments with the final goal of maximizing oral microbiome analyses. Our findings suggest varying optimized sequence numbers according to different study aims: 40 million for microbiota profiling, 50 million for low-abundance species detection, and 20 million for ARG identification. KEY POINTS: • Forty million sequences are a cost-efficient solution for microbiota profiling • Fifty million sequences allow low-abundance species detection • Twenty million sequences are recommended for ARG identification.

Oxford Nanopore sequencing is one of the high-throughput sequencing technologies that facilitates the reconstruction of metagenome-assembled genomes (MAGs). This study aimed to assess the potential of long-read assembly algorithms in Oxford Nanopore sequencing to enhance the MAG-based identification of bacterial pathogens using both simulated and mock communities. Simulated communities were generated to mimic those on fresh spinach and in surface water. Long reads were produced using R9.4.1+SQK-LSK109 and R10.4 + SQK-LSK112, with 0.5, 1, and 2 million reads. The simulated bacterial communities included multidrug-resistant Salmonella enterica serotypes Heidelberg, Montevideo, and Typhimurium in the fresh spinach community individually or in combination, as well as multidrug-resistant Pseudomonas aeruginosa in the surface water community. Real data sets of the ZymoBIOMICS HMW DNA Standard were also studied. A bioinformatic pipeline (MAGenie, freely available at https://github.com/jackchen129/MAGenie) that combines metagenome assembly, taxonomic classification, and sequence extraction was developed to reconstruct draft MAGs from metagenome assemblies. Five assemblers were evaluated based on a series of genomic analyses. Overall, Flye outperformed the other assemblers, followed by Shasta, Raven, and Unicycler, while Canu performed least effectively. In some instances, the extracted sequences resulted in draft MAGs and provided the locations and structures of antimicrobial resistance genes and mobile genetic elements. Our study showcases the viability of utilizing the extracted sequences for precise phylogenetic inference, as demonstrated by the consistent alignment of phylogenetic topology between the reference genome and the extracted sequences. R9.4.1+SQK-LSK109 was more effective in most cases than R10.4+SQK-LSK112, and greater sequencing depths generally led to more accurate results.IMPORTANCEBy examining diverse bacterial communities, particularly those housing multiple Salmonella enterica serotypes, this study holds significance in uncovering the potential of long-read assembly algorithms to improve metagenome-assembled genome (MAG)-based pathogen identification through Oxford Nanopore sequencing. Our research demonstrates that long-read assembly stands out as a promising avenue for boosting precision in MAG-based pathogen identification, thus advancing the development of more robust surveillance measures. The findings also support ongoing endeavors to fine-tune a bioinformatic pipeline for accurate pathogen identification within complex metagenomic samples.

OBJECTIVE: To evaluate non-invasive prenatal testing (NIPT) and expanded non-invasive prenatal testing (NIPT-plus) for detecting aneuploidies at different sequencing depths and assess Z-score accuracy in predicting trisomies 21, 18, 13, 45X, and 47XXX.
METHODS: Pregnancies with positive NIPT or NIPT-plus results detected at the prenatal diagnosis center of Nanfang Hospital were included in this retrospective study, between January 2017 and December 2022. Invasive prenatal diagnostic results were collected. Logistic regression analyses were used to study the relationship between Z-score and positive predictive value (PPV). Optimal cut-off values were obtained based on receiver operating characteristic analysis, and PPVs were calculated in different groups.
RESULTS: We evaluated 1348 pregnant women with positive results, including 930 reported by NIPT and 418 reported by NIPT-plus. NIPT reported significantly more rare chromosomal aneuploidies (RCAs), and NIPT-plus had a significantly higher PPV for trisomy 21 (T21). Logistic regression analyses showed a significant association (P < 0.001) between Z-score and PPVs for T21 and trisomy 18 (T18). A linear relationship was observed between fetal fraction (FF) and Z-values in the true positive cases of T21 and T18.The high Z-score group had significantly higher PPVs than the low Z-score group for T21, T18, trisomy 13, and 47XXX, but not for 45X.
CONCLUSIONS: The Z-score is helpful in assessing NIPT or NIPT-plus results. Therefore, we suggest including the Z-score and FF in the results. By combining the Z-score, FF, and maternal age, clinicians can interpret NIPT results more accurately and improve personal counsel to reduce patients\' anxiety.

BACKGROUND: Parameters adversely affecting the contiguity and accuracy of the assemblies from Illumina next-generation sequencing (NGS) are well described. However, past studies generally focused on their additive effects, overlooking their potential interactions possibly exacerbating one another\'s effects in a multiplicative manner. To investigate whether or not they act interactively on de novo genome assembly quality, we simulated sequencing data for 13 bacterial reference genomes, with varying levels of error rate, sequencing depth, PCR and optical duplicate ratios.
RESULTS: We assessed the quality of assemblies from the simulated sequencing data with a number of contiguity and accuracy metrics, which we used to quantify both additive and multiplicative effects of the four parameters. We found that the tested parameters are engaged in complex interactions, exerting multiplicative, rather than additive, effects on assembly quality. Also, the ratio of non-repeated regions and GC% of the original genomes can shape how the four parameters affect assembly quality.
CONCLUSIONS: We provide a framework for consideration in future studies using de novo genome assembly of bacterial genomes, e.g. in choosing the optimal sequencing depth, balancing between its positive effect on contiguity and negative effect on accuracy due to its interaction with error rate. Furthermore, the properties of the genomes to be sequenced also should be taken into account, as they might influence the effects of error sources themselves.

Limiting harm to organisms caused by genetic sampling is an important consideration for rare species, and a number of non-destructive sampling techniques have been developed to address this issue in freshwater mussels. Two methods, visceral swabbing and tissue biopsies, have proven to be effective for DNA sampling, though it is unclear as to which method is preferable for genotyping-by-sequencing (GBS). Tissue biopsies may cause undue stress and damage to organisms, while visceral swabbing potentially reduces the chance of such harm. Our study compared the efficacy of these two DNA sampling methods for generating GBS data for the unionid freshwater mussel, the Texas pigtoe (Fusconaia askewi). Our results find both methods generate quality sequence data, though some considerations are in order. Tissue biopsies produced significantly higher DNA concentrations and larger numbers of reads when compared with swabs, though there was no significant association between starting DNA concentration and number of reads generated. Swabbing produced greater sequence depth (more reads per sequence), while tissue biopsies revealed greater coverage across the genome (at lower sequence depth). Patterns of genomic variation as characterized in principal component analyses were similar regardless of the sampling method, suggesting that the less invasive swabbing is a viable option for producing quality GBS data in these organisms.

Next-generation sequencing (NGS) has raised a growing interest in phage display research. Sequencing depth is a pivotal parameter for using NGS. In the current study, we made a side-by-side comparison of two NGS platforms with different sequencing depths, denoted as lower-throughput (LTP) and higher-throughput (HTP). The capacity of these platforms for characterization of the composition, quality, and diversity of the unselected Ph.D.TM-12 Phage Display Peptide Library was investigated. Our results indicated that HTP sequencing detects a considerably higher number of unique sequences compared to the LTP platform, thus covering a broader diversity of the library. We found a larger percentage of singletons, a smaller percentage of repeated sequences, and a greater percentage of distinct sequences in the LTP datasets. These parameters suggest a higher library quality, resulting in potentially misleading information when using LTP sequencing for such assessment. Our observations showed that HTP reveals a broader distribution of peptide frequencies, thus revealing increased heterogeneity of the library by the HTP approach and offering a comparatively higher capacity for distinguishing peptides from each other. Our analyses suggested that LTP and HTP datasets show discrepancies in their peptide composition and position-specific distribution of amino acids within the library. Taken together, these findings lead us to the conclusion that a higher sequencing depth can yield more in-depth insights into the composition of the library and provide a more complete picture of the quality and diversity of phage display peptide libraries.

Over the last decade, the field of systems vaccinology has emerged, in which high throughput transcriptomics and other omics assays are used to probe changes of the innate and adaptive immune system in response to vaccination. The goal of this study was to benchmark key technical and analytical parameters of RNA sequencing (RNA-seq) in the context of a multi-site, double-blind randomized vaccine clinical trial.
We collected longitudinal peripheral blood mononuclear cell (PBMC) samples from 10 subjects before and after vaccination with a live attenuated Francisella tularensis vaccine and performed RNA-Seq at two different sites using aliquots from the same sample to generate two replicate datasets (5 time points for 50 samples each). We evaluated the impact of (i) filtering lowly-expressed genes, (ii) using external RNA controls, (iii) fold change and false discovery rate (FDR) filtering, (iv) read length, and (v) sequencing depth on differential expressed genes (DEGs) concordance between replicate datasets. Using synthetic mRNA spike-ins, we developed a method for empirically establishing minimal read-count thresholds for maintaining fold change accuracy on a per-experiment basis. We defined a reference PBMC transcriptome by pooling sequence data and established the impact of sequencing depth and gene filtering on transcriptome representation. Lastly, we modeled statistical power to detect DEGs for a range of sample sizes, effect sizes, and sequencing depths.
Our results showed that (i) filtering lowly-expressed genes is recommended to improve fold-change accuracy and inter-site agreement, if possible guided by mRNA spike-ins (ii) read length did not have a major impact on DEG detection, (iii) applying fold-change cutoffs for DEG detection reduced inter-set agreement and should be used with caution, if at all, (iv) reduction in sequencing depth had a minimal impact on statistical power but reduced the identifiable fraction of the PBMC transcriptome, (v) after sample size, effect size (i.e. the magnitude of fold change) was the most important driver of statistical power to detect DEG. The results from this study provide RNA sequencing benchmarks and guidelines for planning future similar vaccine studies.