Scaling up DNA metabarcoding for freshwater macrozoobenthos monitoring

The viability of DNA metabarcoding for assessment of freshwater macrozoobenthos has been demonstrated over the past years. It matured to a stage where it can be applied to monitoring at a large scale, keeping pace with increased high throughput sequencing (HTS) capacity. However, workflows and sample tagging need to be optimized to accommodate for hundreds of samples within a single sequencing run. We here conceptualize a streamlined metabarcoding workflow, in which samples are processed in 96-well plates. Each sample is replicated starting with tissue extraction. Negative and positive controls are included to ensure data reliability. With our newly developed fusion primer sets for the BF2+BR2 primer pair up to three 96-well plates (288 wells) can be uniquely tagged for a single Illumina sequencing run. By including Illumina indices, tagging can be extended to thousands of samples. We hope that our metabarcoding workflow will be used as a practical guide for future large-scale biodiversity assessments involving freshwater invertebrates. However, we also want to point out that this is just one possible metabarcoding approach, and that we hope this article will stimulate discussion and publication of alternatives and extensions.

Scaling up DNA metabarcoding for freshwater macrozoobenthos monitoring

The viability of DNA metabarcoding for assessment of freshwater macrozoobenthos has been demonstrated over the past years. It matured to a stage where it can be applied to monitoring at a large scale, keeping pace with increased high throughput sequencing (HTS) capacity. However, workflows and sample tagging need to be optimized to accommodate for hundreds of samples within a single sequencing run. We here conceptualize a streamlined metabarcoding workflow, in which samples are processed in 96-well plates. Each sample is replicated starting with tissue extraction. Negative and positive controls are included to ensure data reliability. With our newly developed fusion primer sets for the BF2+BR2 primer pair up to three 96-well plates (288 wells) can be uniquely tagged for a single Illumina sequencing run. By including Illumina indices, tagging can be extended to thousands of samples. We hope that our metabarcoding workflow will be used as a practical guide for future large-scale biodiversity assessments involving freshwater invertebrates. However, we also want to point out that this is just one possible metabarcoding approach, and that we hope this article will stimulate discussion and publication of alternatives and extensions.

Scaling up DNA metabarcoding for freshwater macrozoobenthos monitoring

The viability of DNA metabarcoding for assessment of freshwater macrozoobenthos has been demonstrated over the past years. It matured to a stage where it can be applied to monitoring at a large scale, keeping pace with increased high throughput sequencing (HTS) capacity. However, workflows and sample tagging need to be optimized to accommodate for hundreds of samples within a single sequencing run. We here conceptualize a streamlined metabarcoding workflow, in which samples are processed in 96-well plates. Each sample is replicated starting with tissue extraction. Negative and positive controls are included to ensure data reliability. With our newly developed fusion primer sets for the BF2+BR2 primer pair up to three 96-well plates (288 wells) can be uniquely tagged for a single Illumina sequencing run. By including Illumina indices, tagging can be extended to thousands of samples. We hope that our metabarcoding workflow will be used as a practical guide for future large-scale biodiversity assessments involving freshwater invertebrates. However, we also want to point out that this is just one possible metabarcoding approach, and that we hope this article will stimulate discussion and publication of alternatives and extensions.

COMPARATIVE ANALYSIS OF THE EFICENCY OF POWERPLEX 16 AND POWERPLEX FUSION MULTIPLEX STR KITS IN THE ANALYSIS OF THE CHALLENGING FORENSIC SAMPLES

Since the introduction of the term low copy number DNA, also referred as low template DNA, touch DNA or trace DNA analysis, it has quickly become focal point of forensic DNA testing as well as other DNA based studies. Low template DNA (ltDNA) samples can be described as the samples which involve single source samples with template DNA in concentrations below 100 picograms (pg). Due to sensitivity of ltDNA samples to contamination, it is of great importance to optimize performance of the multiplex STR systems and existing protocols to increase chance of successful analysis. The main objective of this study was analysis of 20 challenging samples (skeletal remains, cigarette buts, chewing gum, poorly collected buccal swabs etc.) mostly low template DNA samples, preliminarily profiled by PowerPlex® 16 multiplex STR systems and additionally processed with new generation multiplex STR kit PowerPlex® Fusion. Sample isolation was done using a standard phenol-chloroform method for bone samples and DNeasy® Blood and Tissue Kit for other forensic samples. PowerPlex® 16 (PP16), multiplex STR system and PowerPlex® Fusion (PP Fusion) were used for co-amplification of 15 and 24 autosomal STR loci respectively. Results of this preliminary study suggest that PP Fusion primer set is better optimized for the analysis of ltDNA samples, and it is more robust regarding presence of the potential PCR inhibitors.

Scaling up DNA metabarcoding for freshwater macrozoobenthos monitoring

The viability of DNA metabarcoding for assessment of freshwater macrozoobenthos has been demonstrated over the past years. It matured to a stage where it can be applied to monitoring at a large scale, keeping pace with increased high throughput sequencing (HTS) capacity. However, workflows and sample tagging need to be optimized to accommodate for hundreds of samples within a single sequencing run. We here conceptualize a streamlined metabarcoding workflow, in which samples are processed in 96-well plates. Each sample is replicated starting with tissue extraction. Negative and positive controls are included to ensure data reliability. With our newly developed fusion primer sets for the BF2+BR2 primer pair up to three 96-well plates (288 wells) can be uniquely tagged for a single Illumina sequencing run. By including Illumina indices tagging can be extended to thousands of samples. We hope that our metabarcoding workflow will be used as a practical guide for future large-scale biodiversity assessments involving freshwater invertebrates. However, we also want to point out that this is just one approach, and that we hope this article will stimulate discussion and publication of alternatives and extensions.

Scaling up DNA metabarcoding for freshwater macrozoobenthos monitoring

The viability of DNA metabarcoding for assessment of freshwater macrozoobenthos has been demonstrated over the past years. It matured to a stage where it can be applied to monitoring at a large scale, keeping pace with increased high throughput sequencing (HTS) capacity. However, workflows and sample tagging need to be optimized to accommodate for hundreds of samples within a single sequencing run. We here conceptualize a streamlined metabarcoding workflow, in which samples are processed in 96-well plates. Each sample is replicated starting with tissue extraction. Negative and positive controls are included to ensure data reliability. With our newly developed fusion primer sets for the BF2+BR2 primer pair up to three 96-well plates (288 wells) can be uniquely tagged for a single Illumina sequencing run. By including Illumina indices tagging can be extended to thousands of samples. We hope that our metabarcoding workflow will be used as a practical guide for future large-scale biodiversity assessments involving freshwater invertebrates. However, we also want to point out that this is just one approach, and that we hope this article will stimulate discussion and publication of alternatives and extensions.

Scaling up freshwater macrozoobenthos metabarcoding with new BF2+BR2 fusion primer sets in 96-well format

The viability of DNA metabarcoding for assessment of freshwater macrozoobenthos has been demonstrated over the past years. It matured to a stage where it can be applied to monitoring at a large scale, keeping pace with increased high throughput sequencing (HTS) capacity. However, workflows and sample tagging need to be optimized to accommodate for hundreds of samples within a single sequencing run. We here conceptualize a streamlined metabarcoding workflow, in which samples are processed in 96-well plates. Each sample is replicated starting with tissue extraction. Negative and positive controls are included to ensure data reliability. With our newly developed fusion primer sets for the BF2+BR2 primer pair up to three 96-well plates (288 samples) can be uniquely tagged for a single Illumina sequencing run. By including Illumina indices tagging can by extended to thousands of samples. We hope that our metabarcoding workflow will be used as practical guide for future large-scale biodiversity assessments involving freshwater invertebrates. However, we also want to acknowledge that this is just one approach, and that we hope this article will stimulate discussion and publication of alternatives and extensions.

Quantitative Amplification of Cleaved Ends (qACE) to assay miRNA-directed target cleavage

microRNA (miRNA) regulation is crucial to achieve precise spatio-temporal expression patterns of their target genes. This makes it crucial to determine the levels of cleavage of a particular target mRNA in different tissues and under different conditions. We developed a quantitative PCR method “quantitative Amplification of Cleaved Ends (qACE)” to assay levels of specific cleavage products in order to determine the extent of miRNA-directed target cleavage of a specific target gene. qACE uses cDNA generated from adapter-ligated RNA molecules and relies on a carefully designed fusion primer that spans the adapter-cleaved RNA junction in qPCR to specifically amplify and quantify cleaved products. The levels of full-length transcripts can also be assayed in the same cDNA preparation using primers that span across the miRNA cleavage site. We used qACE to demonstrate that soybean roots over-expressing miR164 had increased levels of target cleavage and that miRNA deficient Arabidopsis thaliana hen1-1 mutants had reduced levels of target cleavage. We used qACE to discover that differential cleavage by miR164 in nodule vs. adjacent root tissue contributed to nodule-specific expression of NAC1 transcription factors in soybean. These experiments show that qACE can be used to discover and demonstrate tissue-specific cleavage by miRNAs to achieve specific spatio-temporal expression of target genes in plants.