DNA metabarcoding of insects and allies: an evaluation of primers and pipelines

Abstract

Metabarcoding, the coupling of DNA-based species identification and high-throughput sequencing, offers enormous promise for arthropod biodiversity studies but factors such as cost, speed and ease-of-use of bioinformatic pipelines, crucial for making the leapt from demonstration studies to a real-world application, have not yet been adequately addressed. Here, four published and one newly designed primer sets were tested across a diverse set of 80 arthropod species, representing 11 orders, to establish optimal protocols for Illumina-based metabarcoding of tropical Malaise trap samples. Two primer sets which showed the highest amplification success with individual specimen polymerase chain reaction (PCR, 98%) were used for bulk PCR and Illumina MiSeq sequencing. The sequencing outputs were subjected to both manual and simple metagenomics quality control and filtering pipelines. We obtained acceptable detection rates after bulk PCR and high-throughput sequencing (80-90% of input species) but analyses were complicated by putative heteroplasmic sequences and contamination. The manual pipeline produced similar or better outputs to the simple metagenomics pipeline (1.4 compared with 0.5 expected:unexpected Operational Taxonomic Units). Our study suggests that metabarcoding is slowly becoming as cheap, fast and easy as conventional DNA barcoding, and that Malaise trap metabarcoding may soon fulfill its potential, providing a thermometer for biodiversity.

Benchmarking DNA Metabarcoding for Biodiversity-Based Monitoring and Assessment

“…Our data corroborate previous studies unveiling the lack of universality in the COI primers, which is translated to biases during PCR step (Pochon et al, 2013;Deagle et al, 2014). However, the increased performance of the short region, previously demonstrated for individual barcoding on marine metazoans (Leray et al, 2013) and metabarcoding in insects (Brandon-Mong et al, 2015) proves that the mlCOI barcode retrieves a high proportion of the morphologically identified taxa. This fact also corroborates the preferred use of small barcodes for metabarcoding, which provide pair-end overlaps on Illumina sequencing and good taxonomic resolution for species identification (Meusnier et al, 2008).…”

Validation and Development of COI Metabarcoding Primers for Freshwater Macroinvertebrate Bioassessment

“…However, the COI barcoding gene region shows high codon degeneracy throughout its sequence, making the design of such "truly" universal primers difficult (Deagle et al, 2014;Sharma and Kobayashi, 2014). Several COI barcoding primers with different levels of base degeneracy have been developed of which many are now used or could be suitable for metabarcoding studies (Figure 1, e.g., Folmer et al, 1994;Hebert et al, 2004;Meusnier et al, 2008;Van Houdt et al, 2010;Shokralla et al, 2011Shokralla et al, , 2015Zeale et al, 2011;Geller et al, 2013;Leray et al, 2013;Gibson et al, 2014;Brandon-Mong et al, 2015). However, often these primers were developed for a specific taxonomic group, purpose or ecosystem, for example the primers by Zeale et al (2011) which were originally developed for gut content analysis on bats but are now more widely used.…”

“…However, often these primers were developed for a specific taxonomic group, purpose or ecosystem, for example the primers by Zeale et al (2011) which were originally developed for gut content analysis on bats but are now more widely used. Thus, despite including several degenerate bases, metabarcoding primers typically recover only 80-90% or even less of the taxa present in a sample (Leray et al, 2013;Brandon-Mong et al, 2015;Elbrecht and Leese, 2015). Furthermore, many primers have not been thoroughly evaluated for primer bias and the proportion of undetected taxa, making development and testing of universal primers a pressing issue.…”

“…Additionally, details on criteria for primer design such as the used reference sequence data are often not described extensively (e.g., Hajibabaei et al, 2011;Shokralla et al, 2015). Typically, primers are developed either with aligned reference barcode sequences for the taxonomic target groups available from NCBI or BOLD (Zeale et al, 2011;Leray et al, 2013;Gibson et al, 2014), or alternatively only mitochondrial genomes or a small subset of barcoding sequences are used (Geller et al, 2013;Deagle et al, 2014;Brandon-Mong et al, 2015). These two approaches are typically biased, as sequences for certain taxa are overrepresented in big datasets (e.g., from population genetic studies), while datasets containing only mitochondrial genomes 1 100 200 300 400 500 600 658 FIGURE 1 | Selection of potential COI primer sets for DNA metabarcoding of insects, targeting the Folmer region.…”

Field calibration of blowfly-derived DNA against traditional methods for assessing mammal diversity in tropical forests

“…Reads with mean scores of less than 5 (sliding window size = 5, step size = 5) or minimum tail length of poly-A/T tails of five at 5= end and five at 3= end were trimmed of low-quality bases at 5= end (1 bp) and 3= end (1 bp). The output data (in FASTA format) was then further filtered and trimmed of primer sequences with quality control and filtering steps performed manually in CodonCode Aligner (CondonCode Corp., USA) following Brandon-Mong et al (2015). Both consensus and singleton reads surviving the quality control and filtering steps were retained for taxonomic assignment.…”