Optimization and Application of direct PCR in community metabarcoding

Direct PCR allows the amplification of DNA from animal or plant tissue samples without need for extraction and purification steps. For this procedure, dry is homogenized, dissolved in water subsequently amplified, thus, its successful application largely depends on absence inhibitors. Although method has been successfully applied barcoding approaches invertebrates, it not yet attempted metabarcoding approaches. We used nonbiting midges (Diptera: Chironomidae) to test if amplicons produced by direct could be next-generation sequencing. To access whether applicable a variety chironomid species, we tested 236 adult specimens randomly selected emergence traps an artificial pond mesocosm. ground tissue, corresponding 0.1% specimens’ biomass, protocol following Wong et al. (2014) amplification. In total, 98 % were amplified found diverse community comprised 20 different genera. order compare ´traditional´ isolation-PCR, created mock communities (14 species) both 421 bp COI fragment. After second indexing adapter ligation, sequenced Illumina sequencer. only slightly lower recovery rates with approach compared traditional protocols. These further improved methods when equal biomass (ca. 0.006 mg) was used. With our approach, possible detect species which constituted 1% entire sample. Generally, did have large effect sequence read abundance. However, abundance varied strongly between species. are currently investigating caused primer bias artifact differently degraded tissue. This study proof principle that can sequencing, applications future biomonitoring projects portable laboratory technologies. using technique monitor large-scale experiment (artificial mesocosm facility) covering weekly taken over two summer half-years.