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1. Quantification of Circulating Cell Free Mitochondrial DNA in Extracellular Vesicles with PicoGreen™ in Liquid Biopsies: Fast Assessment of Disease/Trauma Severity

   https://doi.org/10.3390/cells10040819  

   Marcatti, Michela; Saada, Jamal; Okereke, Ikenna; Wade, Charles E.; Bossmann, Stefan H.; Motamedi, Massoud; Szczesny, Bartosz (April 2021, Cells)

   null (Ed.)

   The analysis of circulating cell free DNA (ccf-DNA) is an emerging diagnostic tool for the detection and monitoring of tissue injury, disease progression, and potential treatment effects. Currently, most of ccf-DNA in tissue and liquid biopsies is analysed with real-time quantitative PCR (qPCR) that is primer- and template-specific, labour intensive and cost-inefficient. In this report we directly compare the amounts of ccf-DNA in serum of healthy volunteers, and subjects presenting with various stages of lung adenocarcinoma, and survivors of traumatic brain injury using qPCR and quantitative PicoGreen™ fluorescence assay. A significant increase of ccf-DNA in lung adenocarcinoma and traumatic brain injury patients, in comparison to the group of healthy human subjects, was found using both analytical methods. However, the direct correlation between PicoGreen™ fluorescence and qPCR was found only when mitochondrial DNA (mtDNA)-specific primers were used. Further analysis of the location of ccf-DNA indicated that the majority of DNA is located within lumen of extracellular vesicles (EVs) and is easily detected with mtDNA-specific primers. We have concluded that due to the presence of active DNases in the blood, the analysis of DNA within EVs has the potential of providing rapid diagnostic outcomes. Moreover, we speculate that accurate and rapid quantification of ccf-DNA with PicoGreen™ fluorescent probe used as a point of care approach could facilitate immediate assessment and treatment of critically ill patients. 

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2. Leveraging whole‐genome sequencing to estimate telomere length in plants

   https://doi.org/10.1111/1755-0998.13899  

   Zavala‐Paez, Michelle; Holliday, Jason; Hamilton, Jill A. (November 2023, Molecular Ecology Resources)

   Abstract Changes in telomere length are increasingly used to indicate species' response to environmental stress across diverse taxa. Despite this broad use, few studies have explored telomere length in plants. Thus, evaluation of new approaches for measuring telomeres in plants is needed. Rapid advances in sequencing approaches and bioinformatic tools now allow estimation of telomere content from whole‐genome sequencing (WGS) data, a proxy for telomere length. While telomere content has been quantified extensively using quantitative polymerase chain reaction (qPCR) and WGS in humans, no study to date has compared the effectiveness of WGS in estimating telomere length in plants relative to qPCR approaches. In this study, we use 100Populusclones re‐sequenced using short‐read Illumina sequencing to quantify telomere length comparing three different bioinformatic approaches (Computel, K‐seek and TRIP) in addition to qPCR. Overall, telomere length estimates varied across different bioinformatic approaches, but were highly correlated across methods for individual genotypes. A positive correlation was observed between WGS estimates and qPCR, however, Computel estimates exhibited the greatest correlation. Computel incorporates genome coverage into telomere length calculations, suggesting that genome coverage is likely important to telomere length quantification when using WGS data. Overall, telomere estimates from WGS provided greater precision and accuracy of telomere length estimates relative to qPCR. The findings suggest WGS is a promising approach for assessing telomere length and, as the field of telomere ecology evolves, may provide added value to assaying response to biotic and abiotic environments for plants needed to accelerate plant breeding and conservation management. 

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3. A Two-Step PCR Protocol Enabling Flexible Primer Choice and High Sequencing Yield for Illumina MiSeq Meta-Barcoding

   https://doi.org/10.3390/agronomy11071274  

   Chen, Ko-Hsuan; Longley, Reid; Bonito, Gregory; Liao, Hui-Ling (July 2021, Agronomy)

   null (Ed.)

   High-throughput amplicon sequencing that primarily targets the 16S ribosomal DNA (rDNA) (for bacteria and archaea) and the Internal Transcribed Spacer rDNA (for fungi) have facilitated microbial community discovery across diverse environments. A three-step PCR that utilizes flexible primer choices to construct the library for Illumina amplicon sequencing has been applied to several studies in forest and agricultural systems. The three-step PCR protocol, while producing high-quality reads, often yields a large number (up to 46%) of reads that are unable to be assigned to a specific sample according to its barcode. Here, we improve this technique through an optimized two-step PCR protocol. We tested and compared the improved two-step PCR meta-barcoding protocol against the three-step PCR protocol using four different primer pairs (fungal ITS: ITS1F-ITS2 and ITS1F-ITS4, and bacterial 16S: 515F-806R and 341F-806R). We demonstrate that the sequence quantity and recovery rate were significantly improved with the two-step PCR approach (fourfold more read counts per sample; determined reads ≈90% per run) while retaining high read quality (Q30 > 80%). Given that synthetic barcodes are incorporated independently from any specific primers, this two-step PCR protocol can be broadly adapted to different genomic regions and organisms of scientific interest. 

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4. Widely used, short 16S rRNA mitochondrial gene fragments yield poor and erratic results in phylogenetic estimation and species delimitation of amphibians

   https://doi.org/10.1186/s12862-022-01994-y  

   Chan, Kin Onn; Hertwig, Stefan T.; Neokleous, Dario N.; Flury, Jana M.; Brown, Rafe M. (December 2022, BMC Ecology and Evolution)

   Abstract Background The 16S mitochondrial rRNA gene is the most widely sequenced molecular marker in amphibian systematic studies, making it comparable to the universal CO1 barcode that is more commonly used in other animal groups. However, studies employ different primer combinations that target different lengths/regions of the 16S gene ranging from complete gene sequences (~ 1500 bp) to short fragments (~ 500 bp), the latter of which is the most ubiquitously used. Sequences of different lengths are often concatenated, compared, and/or jointly analyzed to infer phylogenetic relationships, estimate genetic divergence ( p -distances), and justify the recognition of new species (species delimitation), making the 16S gene region, by far, the most influential molecular marker in amphibian systematics. Despite their ubiquitous and multifarious use, no studies have ever been conducted to evaluate the congruence and performance among the different fragment lengths. Results Using empirical data derived from both Sanger-based and genomic approaches, we show that full-length 16S sequences recover the most accurate phylogenetic relationships, highest branch support, lowest variation in genetic distances (pairwise p -distances), and best-scoring species delimitation partitions. In contrast, widely used short fragments produce inaccurate phylogenetic reconstructions, lower and more variable branch support, erratic genetic distances, and low-scoring species delimitation partitions, the numbers of which are vastly overestimated. The relatively poor performance of short 16S fragments is likely due to insufficient phylogenetic information content. Conclusions Taken together, our results demonstrate that short 16S fragments are unable to match the efficacy achieved by full-length sequences in terms of topological accuracy, heuristic branch support, genetic divergences, and species delimitation partitions, and thus, phylogenetic and taxonomic inferences that are predicated on short 16S fragments should be interpreted with caution. However, short 16S fragments can still be useful for species identification, rapid assessments, or definitively coupling complex life stages in natural history studies and faunal inventories. While the full 16S sequence performs best, it requires the use of several primer pairs that increases cost, time, and effort. As a compromise, our results demonstrate that practitioners should utilize medium-length primers in favor of the short-fragment primers because they have the potential to markedly improve phylogenetic inference and species delimitation without additional cost. 

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5. Introducing a novel 28S rRNA marker for improved assessment of coral reef biodiversity

   https://doi.org/10.22541/au.172529899.94609833/v1  

   Martineau, Gabrielle; Toonen, Robert; Timmers, Molly; Jury, Christopher; Vicente, Jan (September 2024, Authorea Inc.)

   Biodiversity monitoring based on DNA metabarcoding depends on primer performance. Here, we develop a new metabarcoding primer pair that targets a ~ 318 bp fragment of the 28S rRNA gene. We validate the primer pair in assessing sponges, a notoriously challenging group for coral reef metabarcoding studies, by using mock and natural complex reef communities to examine its performance in species detection, amplification efficiency, and quantitative potential. Mock community experiments revealed a high number of sponge species (n = 94) spanning a broad taxonomic scope (15 orders), limited taxon-specific primer biases (only a single species exceeded a two-fold deviation from the expected number of reads), and its suitability for quantitative metabarcoding – there was a significant relationship between read abundance and visual percent coverage of sponge taxa (R = 0.76). In the natural complex coral reef community experiments, commonly used COI metabarcoding primers detected only 30.9% of sponge species, while the new 28S primer increased detection to 79.4%. These new 28S primers detect a broader taxonomic array of species across phyla and classes within the complex cryptobiome of coral reef communities than the Leray-Geller COI primers. As biodiversity assessments using metabarcoding tools are increasingly being leveraged for environmental monitoring and guide policymaking, these new 28S rRNA primers can improve biodiversity assessments for complex ecological coral reef communities. 

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