Scaling up freshwater macrozoobenthos metabarcoding with new BF2+BR2 fusion primer sets in 96-well format
- Published
- Accepted
- Subject Areas
- Molecular Biology, Freshwater Biology
- Keywords
- high throughput sequencing, macrozoobenthos, multiplexing, Biomonitoring, metabarcoding, fusion primers, tagging, illumina, DNAqua.net, replication
- Copyright
- © 2017 Elbrecht et al.
- Licence
- This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ Preprints) and either DOI or URL of the article must be cited.
- Cite this article
- 2017. Scaling up freshwater macrozoobenthos metabarcoding with new BF2+BR2 fusion primer sets in 96-well format. PeerJ Preprints 5:e3456v1 https://doi.org/10.7287/peerj.preprints.3456v1
Abstract
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.
Author Comment
initial manuscript version to be published at mbmg
Supplemental Information
Figure S1: Newly developed fusion primer sets (BF2+BR2), suitable for tagging 288 individual wells
Figure S2: Base composition of the inline tagging region
Figure S3: Similarity between inline barcodes across all fusion primers
Script S1: R script used for randomly generating inline barcodes for the given primer sets (includes visualization, as shown in Figure S3 and Figure 3)
Table S1: Table providing an overview of proposed tagging combinations (as shown in Figure 3)
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