title: Illumina NovaSeq 6000 paired end sequencing
design: DNA extracted and purified from a bulk insect sample that was homogenized (insect soup) as described in Persson et al 2024: dx.doi.org/10.17504/protocols.io.yxmvm31bbl3p/v1. For amplicon library preparation we use a two-step PCR approach. In the first step (PCR 1) we amplify the 2 target genes in independent reactions: 418 base pairs of the mitochondrial cytochrome oxidase 1 (CO1) of arthropods and ~253bp of the bacterial 16S rRNA gene, V4 hypervariable region. In the second step (PCR 2) we add the indexes to the amplified sequences and fill up Illumina adapters. We used a unique forward and a unique reverse index for each sample , but the same two indexes were used for the mtDNA COI and bacterial 16S rRNA products of the same sample. That way, upon pooling, the separately-prepared amplicons for the two target regions of the same sample effectively became a single library and were, therefore, sequenced together.
library_name: P28409_1121
library_construction_protocol: DNA extracted and purified from a bulk insect sample that was homogenized (insect soup) as described in Persson et al 2024: dx.doi.org/10.17504/protocols.io.yxmvm31bbl3p/v1. Homogenate aliquots (225 ul) were 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 for amplicon library preparation using a two-step PCR approach. In the first step (PCR 1) we amplify the two target genes in independent reactions: 418 base pairs of the mitochondrial cytochrome oxidase 1 (CO1) of arthropods and ~253bp of the bacterial 16S rRNA gene, V4 hypervariable region. 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 were added and Illumina adapters completed. We used a unique forward and a unique reverse index for each sample, but the same two indexes were used for the mtDNA COI and bacterial 16S rRNA products of the same sample. That way, upon pooling, the separately-prepared amplicons for the two target regions of the same sample effectively became a single library and were, therefore, sequenced together. Previously to pooling the we assessed the concentration of PCR II products on an agarose gel based on the band brightness. All samples were pooled approximately equimolarly to form the sequencing pool which was purified with Promega ProNexï¿½ 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).