Species Identification in Malaise Trap Samples by DNA Barcoding Based on NGS Technologies and a Scoring Matrix

The German Barcoding initiatives BFB and GBOL have generated a reference library of more than 16,000 metazoan species, which is now ready for applications concerning next generation molecular biodiversity assessments. To streamline the barcoding process, we have developed a meta-barcoding pipeline: We pre-sorted a single malaise trap sample (obtained during one week in August 2014, southern Germany) into 12 arthropod orders and extracted DNA from pooled individuals of each order separately, in order to facilitate DNA extraction and avoid time consuming single specimen selection. Aliquots of each ordinal-level DNA extract were combined to roughly simulate a DNA extract from a non-sorted malaise sample. Each DNA extract was amplified using four primer sets targeting the CO1-5' fragment. The resulting PCR products (150-400bp) were sequenced separately on an Illumina Mi-SEQ platform, resulting in 1.5 million sequences and 5,500 clusters (coverage >10;CD-HIT-EST, 98%). Using a total of 120,000 DNA barcodes of identified, Central European Hymenoptera, Coleoptera, Diptera, and Lepidoptera downloaded from BOLD we established a reference sequence database for a local CUSTOM BLAST. This allowed us to identify 529 Barcode Index Numbers (BINs) from our sequence clusters derived from pooled Malaise trap samples. We introduce a scoring matrix based on the sequence match percentages of each amplicon in order to gain plausibility for each detected BIN, leading to 390 high score BINs in the sorted samples;whereas 268 of these high score BINs (69%) could be identified in the combined sample. The results indicate that a time consuming pre-sorting process will yield approximately 30% more high score BINs compared to the nonsorted sample in our case. These promising results indicate that a fast, efficient and reliable analysis of next generation data from malaise trap samples can be achieved using this pipeline

Phylogeography and population genetics of diving beetles in New Guinea

Predaceous diving beetles (Dytiscidae) are common inhabitants of both lentic and lotic freshwater systems worldwide. They have played a major role in our understanding of the relationship between habitat stability and evolution for dispersal propensity.Numerous endemic diving beetles can be found in on the island of New Guinea. However, like most micro-fauna on remote islands, their evolutionary history and population ecology is largely unknown. In this series of studies, I use both traditional Sanger sequencing and next- generation sequencing techniques to explore the phylogenetic relationship of beetles in a genus as well as the population genetic patterns within a species

Results of the four ordinal level sorted arthropod orders and the combined fraction.

<p>Magnifying glasses represent total number of detected BINs, the Score ≥ 300 symbol represents the total number of detected BINs with a score ≥ 300 within the sample, the CS symbol represents number of shared BINs within the single ordinal level sorted and the combined fraction.</p

Efficiency of amplicons used.

<p>Efficiency of amplicons used.</p

Examples of the scoring scheme used in this study.

<p>The upper Lepidopteran species (<i>Erebia eriphyle</i>, Fryer 1839) was not included into further analyses, as its score summed up to only 220. The lower Hymenopteran species (<i>Megastylus cruentator</i>, Schiødte 1839) represents a “high score BIN” with a total of 1260 points. Gradient code illustrates the used color for percentage values detected for each amplicon.</p

Categories of scoring according to the sequence identity percentage.

<p>Categories of scoring according to the sequence identity percentage.</p

Visualization of the study workflow.

<p>Visualization of the study workflow.</p

Arthropod orders sorted and combined by sample number.

<p>Arthropod orders sorted and combined by sample number.</p

Primer efficiency for CO1 amplification of the four ordinal level sorted arthropod orders.

<p>Primer efficiency for CO1 amplification of the four ordinal level sorted arthropod orders.</p

Primers and corresponding PCR conditions used in this study.

<p>Primers and corresponding PCR conditions used in this study.</p