Illumina NovaSeq 6000 paired end sequencing
DNA was extracted from bulk insect samples that were homogenized (insect soup) as described in Persson et al 2024: dx.doi.org/10.17504/protocols.io.yxmvm31bbl3p/v1. Sample processing consists of wet-weighing the insect sample to obtain insect biomass, adding lysis buffer and proteinase K according to the insect biomass, homogenizing the sample for 2 min at 6000 rpm using a sample grinder (UltraTurrax Tube Drive P control, IKA), incubating the homogenized sample for 2h45m at 56C in a dry shaking incubator and taking a 225 µL aliquot of the homogenate for DNA purification. Homogenate aliquots are then purified using silica-coated magnetic beads with the KingFisher Cell and Tissue DNA kit on a King Fisher Flex 96 robot (both Thermo Scientific) according to manufacturer instructions. Purified DNA was then used as template for the amplification of the target COI fragment via PCR (PCR I). 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. Please note that just before DNA purification we add 2 different synthetic sequences to the homogenate aliquot (see "artificial spikes" attribute).