Schematic overview of the workflows during preparation of artificial chironomid communities. Two different sets of chironomid specimens (Mock A and Mock B) were analysed using different approaches of standard and direct PCR metabarcoding, to study the effect of direct PCR or DNA extraction protocols on detection rates and individual read abundances. Experimental setups included analysing the effects of variable mass per individual and different mock compositions (Experiment 1), variable or similar amounts of different input materials (Experiment 2), and variable concentrations of mock material to assess sensitivity (Experiment 3). Mocks were created by pipetting tissue-water mixes (Mocks A-1, A-2 and B-1), purified DNA extracts (Mock B-2) or PCR products (Mock B-3) of individual specimens. A subsample of Mocks A-1 (a, b) and A-2 (c, d) was used for sequential dilution (Mock A-3). Pie charts indicate if artificial communities were created under a size-sorting scenario (aiming for even masses per individual) or under a non-size-sorting scenario (where individual masses naturally vary). Purified DNA extracts were subjected to PCR, while non-purified tissue-water mixes were used in dPCR approaches. The labels ‘Figure 2’ to ‘Figure 5’ within the figure refer to the corresponding graphical representations of the results.

  Part of: Röder N, Schwenk K (2023) Direct PCR meets high-throughput sequencing – metabarcoding of chironomid communities without DNA extraction. Metabarcoding and Metagenomics 7: e102455.