Next-generation sequencing (NGS) technologies are rapidly increasing the applicability of genomics approaches in biological sciences. Aside from the sheer volume of sequence information obtained from NGS devices, the massively parallelized capacity of these machines is a significant advantage for the analysis of mixtures of DNA fragments. In PCR-based applications of NGS technologies amplicons are generated from target genes, often from multiple template genomes, and are sequenced in parallel without the need for separating target organisms or their DNA through sample sorting or cloning
]. For example, microbial ecologists have taken advantage of NGS technologies in amplicon-based (marker genes) metagenomic studies such as the analysis of 16S rDNA for prokaryotic biodiversity assessments in various ecological settings
]. Similar approaches have been developed for many situations where multi-template (environmental) samples are targets of investigations
] seeks to gain sequence information for standardized species-specific DNA markers—DNA barcodes—directly from environmental samples such as soil, water, air, benthos or gut contents of various organisms
]. This approach has the potential to dramatically increase the applicability of DNA barcoding in situations where rapid assessment of biodiversity at high resolution (e.g. species-level) is required at a wide spatiotemporal scale or in places where access to individual specimens is impossible or impractical. For example, environmental assessment through biomonitoring relies on biodiversity patterns of bioindicator (sentinel) species such as benthic macroinvertebrates, often at larval stage. However, due to difficulties in robust species-level identification of target groups, biomonitoring programs are faced with an identification bottleneck that can then lead to difficulties in implementing these programs
]. We have recently demonstrated the potential of using NGS-based environmental barcoding in identifying species of fresh water benthos
]. This approach has triggered a wholly new biomonitoring paradigm--Biomonitoring 2.0--for environmental assessment
An important concern in the analysis of environmental samples is obtaining DNA templates from all target organisms in the mixed sample. Methods have been developed to extract and purify DNA from environmental samples such as soil or water. However, for bulk material such as benthos--obtained using kick nets--orpassively sampled arthropods collected in a Malaise trap, DNA extraction often requires homogenizing the biomass from all organisms and then performing a standard DNA extraction protocol on this homogenized slurry. Although this approach has been effective in gaining DNA from organisms in the mixture, it results in loss of all individual specimens, thereby rendering any subsequent analysis on these individuals impossible. Recently, we demonstrated that ethanol, commonly used as a preservative medium for storing specimens, contains DNA of stored organism and that this “free DNA” can be directly used for downstream amplification and sequencing without the need for conventional DNA extraction approaches
]. Although we have shown the utility of this approach in individual specimens stored in ethanol, it is not known whether ethanol-based free DNA of many different taxa, with various biomasses, in a mixed environmental sample such as benthos, could be sequenced in an NGS workflow.
Another important concern in the use of NGS for analysis of environmental samples is the issue of bias in multi-template PCR amplification
]. Current workflows often require PCR amplification of target templates from mixed samples, which can result in differential amplification of sequences from some species, leading to qualitative and quantitative biases in sequence representation from target organisms in the mixture. In other words, because of PCR bias, some species may not be amplified and sequenced while others may be amplified and sequenced in excess. This can obscure identity and abundance measures from bulk environmental samples. Although modified amplification regimes have been developed for offsetting the effect of PCR bias
] and methods based on direct sequencing of DNA (without the need for PCR amplification) are on the horizon
], PCR amplification bias remains an important issue. This problem is especially important when environmental samples are used for surveillance and monitoring applications where comparative analysis of biodiversity should be performed objectively and reproducibly
Here we introduce an enhanced approach for environmental barcoding, which will aid sampling, DNA extraction and PCR steps in biodiversity analysis of benthic macroinvertebrate taxa commonly used for biomonitoring applications. We firstly incorporate a non-destructive sample preparation approach by using preservative media (ethanol) as the source of target DNA (hereafter referred to as ethanol-based DNA) and compare it with a conventional tissue-based DNA extraction (hereafter referred to as tissue-DNA). Secondly, to increase the recovery of species’ DNA barcode sequences in bulk environmental samples and to offset specific primer-binding biases, we introduce a multiplex PCR approach targeting multiple amplicons within the standard cytochrome c oxidase 1 (COI) DNA barcode region. We develop and test three wide-range primer sets for NGS analysis. To show the utility of this approach, we test it in parallel with Sanger sequencing individual specimens from a typical biomonitoring benthic sample containing several groups of macroinvertebrates.