Illumina NovaSeq 6000 paired end sequencing
DNA extracted and purified from bulk arthropod sample that was first separated into macrofauna and mesofauna subsamples. Each subsample was independently homogenized with a mortar and pestle followed by DNA extraction of macrofauna and mesofauna subsamples using the Kingfisher Cell and Tissue DNA kit. Before library preparation each sample was reassembled by combining DNA extracts from each subsample pair at a ratio of 1:10 in the amount of DNA of macrofauna to mesofauna. For amplicon library preparation we use a two-step PCR approach. In the first step (PCR 1) we amplify the target gene: 418 base pairs of the� mitochondrial cytochrome oxidase 1 (CO1). In the second step (PCR 2) we add the indexes to the amplified sequences and fill up Illumina adapters. Detailed protocol can be found at https://doi.org/10.1371/journal.pone.0286272
DNA extraction of macrofauna and mesofauna subsamples was performed separately using the Kingfisher Cell and Tissue DNA kit. Specimens were first dried and were then manually homogenized with a pestle and lysed overnight at 56�C by adding 800 �l of lysis buffer and 100 �l of proteinase K (provided in the Kingfisher Cell and Tissue DNA kit). DNA was then purified from a 225 �L aliquot of homogenate using silica-coated magnetic beads with the KingFisher Cell and Tissue DNA kit on a KingFisher Flex 96 robot (both Thermo Scientific) according to manufacturer instructions. DNA extracts were quantified using Qubit� dsDNA HS Assay Kit on a Qubit Fluorometer (INVITROGEN�), and the original sample was reassembled by combining DNA extracts from each subsample pair at a ratio of 1:10 in the amount of DNA of macrofauna to mesofauna. For amplicon library preparation we use a two-step PCR approach. In the first step (PCR 1) we amplify the target gene: 418 base pairs of the� mitochondrial cytochrome oxidase 1 (CO1) using ddH20 as a template. After amplification, PCR products were cleaned using a ratio of 1/2.0 (v/v) of PCR product to magnetic beads. Purified PCR products were then used as template in the second round of PCR (PCR II), where sample-specific tags are added and Illumina adapters completed. The concentration of PCR II products was assessed based on the band brightness on an agarose gel. All samples were pooled approximately equimolarly to form the sequencing pool which was purified with Promega ProNex� Size-Selective Purification System, using 1/1.5 (v/v) pool to magnetic beads ratio. Samples were sequenced on NovaSeq6000 (NovaSeq Control Software 1.8.0/RTA v3.4.4) with a 251nt(Read1)-10nt(Index1)-10nt(Index2)-251nt(Read2) setup using 'NovaSeqXp' workflow in 'SP' mode flowcell. The Bcl to FastQ conversion was performed using bcl2fastq v2.20.0.422 from the CASAVA software suite. The quality scale used is Sanger / phred33 / Illumina 1.8+. Sequencing and demultiplexing was performed at the National Genomic Infrastructure (NGI), SciLifeLab, Stockholm, Sweden. Detailed protocol library preparation can be found in section 4 of protocol dx.doi.org/10.17504/protocols.io.kqdg36261g25/v2