Illumina NovaSeq 6000 paired end sequencing
This is a positive control sample. DNA was extracted and purified from a bulk insect sample using the FAVIS protocol https://doi.org/10.1371/journal.pone.0286272. 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
This is a positive control sample. DNA was extracted from a bulk insect sample that was processed using the Favis protocol: dx.doi.org/10.17504/protocols.io.kqdg36261g25/v2. Sample processing consists of wet-weighing the insect sample to obtain insect biomass, adding lysis buffer according to the insect biomass, incubating the sample with lysis buffer and proteinase K for 2h45m at 56C in a dry shaking incubator, decanting lysates and taking a 225 �L lysate aliquot for DNA purification. Lysate 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. Detailed protocol for sample processing can be found at dx.doi.org/10.17504/protocols.io.kqdg36261g25/v2