Nextera xt indexing kit

Integration site analysis was conducted using MuA-mediated integration site recovery as previously described.⁴³ (link) gDNA was incubated with MuA transposase (Thermo Fisher Scientific) and commercially synthesized annealed oligonucleotides (Data S1, sequence A and B).
MuA digested gDNA was amplified by PCR with a forward primer binding to either super-piggyBac or piggyBat transposon 3′-TIR (Data S1, sequence E), with the generated PCR amplicon library consisting of oligonucleotides spanning the intersection of the 3′ transposon-specific TIR and adjacent gDNA corresponding to genomic integration site. This PCR amplicon library was indexed using Nextera XT Indexing kit (Illumina, San Diego, CA) and purified using JetSeq beads (Bioline, Memphis, TN).
Next-generation sequencing of purified PCR amplicon libraries was performed on the Illumina MiSeq v.3 platform (Australian Genome Research Facility) and mapped to chromosomal loci using previously described bioinformatic algorithms.⁴⁴ (link) Mapped loci were annotated using HOMER hg19 peak enrichment tool,⁴⁵ (link) with frequency of integrations expressed as a log fold change over expected for random insertion. Raw sequencing data have been deposited in the Sequence Read Archive (Sequence Read Archive: PRJNA807916), with all unique identified integration sites provided in Table S1.

PCR products or DNA fragments from agarose gel were purified with the Wizard SV gel and PCR clean-up system (Promega, USA). Plasmid and genomic DNA was isolated using a plasmid minikit and a genomic minikit (A&A Biotechnology, Poland). For DNA manipulation, restriction and modification enzymes purchased from Thermo Fisher Scientific (USA) were used. Samples for genome sequencing were prepared with the Nextera XT DNA sample preparation kit, the Nextera XT indexing kit, and the PhiX control V3 kit (Illumina, USA) according to the manufacturer’s instructions and sequenced on a MiSeq Sequencer (Illumina). The data were analyzed with CLC Genomics Workbench 8.5 (Qiagen, Germany). Nucleotide sequences were translated to corresponding peptide sequences using an online translation tool on the ExPasy server (61 (link)). LiaF and LiaS transmembrane helices were predicted using TMHMM Server v. 2.0 (62 (link)). Conserved domains (CD) and active sites of LiaFSR-X were identified using the CD search online tool at the NCBI Conserved Domain Database (63 (link)). Additionally, LiaX N- and C-terminal domains composed mainly of α-helices and β-strands (22 (link)), respectively, were predicted based on the structure modeled using the I-TASSER web service (64 (link)). Protein models were visualized using Protter (65 (link)).

We purified the resulting amplicons to remove primer dimers and fragments less than 100bp by binding PCR products to 20uL of Serapure beads [47 (link)] and performing ethanol washes. DNA was subsequently eluted from the beads with 50uL of 10mM Tris pH 8.5. Products were again visualized on a gel to ensure removal of small fragments. Following PCR purification, we prepared libraries for sequencing by attaching unique dual index combinations to DNA from each individual using the Nextera XT indexing kit (Illumina, San Diego, CA), following the manufacturer’s protocol. We then purified the indexed amplicons using Serapure as before. Indexed amplicons were then quantified using a Qubit dsDNA BR Assay (Life Technologies, Carlsbad, CA), visualized using an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA) and then pooled in equimolar ratios for 2x250 sequencing on an Illumina MiSeq machine (Illumina, San Diego, CA) at the Technology Center for Genomics and Bioinformatics (University of California, Los Angeles).

To determine the community composition of microarthropods (mites: Acari, and springtails: Collembola), the large intact soil core was extracted using the Tullgren extractors at Lancaster University. Per plot, the batch of extracted microarthropods was collected in 96% ethanol and further processed to enable DNA-based identification. DNA extraction was performed using the DNeasy Blood & Tissue kit. DNA was amplified using the MiteMinBarF7 and MiteMinBarR4 primers, which target an approximately 200 bp fragment located within the cytochrome oxidase subunit 1 (COI) region, and were specifically designed to cover a wide diversity of microarthropods in NW-European grasslands [42 ]. At Aarhus University (Roskilde, Denmark), amplicons were prepared for in-house paired-end sequencing on an Illumina MiSeq platform, using the Nextera XT indexing kit (Illumina, San Diego, CA, USA). The resulting amplicon libraries were purified using HighPrep PCR (Magbio Genomics Inc., Gaithersburg, MD, USA) beads, quantified and equimolarly pooled, upon sequencing using the 250 bp paired-end MiSeq version 2 reagent kit (Illumina, San Diego, CA, USA).

For each accession, libraries were constructed and barcoded using the Nextera XT library preparation kit and the Nextera XT indexing kit (Illumina) following the instructions of the manufacturer with minor modifications. For the library preparation, only 25% of the recommended amount of reagents were used, and the libraries were purified using the Agencourt AMPure system (Beckman Coulter). Libraries were quantified by performing the dsDNA HS assay on a Qubit system (Invitrogen, Thermo Fisher Scientific). Library quality was further assessed by running 5 µl on a 1.5% agarose gel (40 min, 100 V). Only libraries for which the majority of fragments was at least 300 bp in length were processed further. Libraries were pooled to equal concentrations (ng/µL) and sequenced on a MiSeq (Illumina) at the Georgia Genomics Facility (University of Georgia).

Bacterial 16S V3-V4 regions were amplified by PCR using universal primers derived from DBact-0341-b-S-17 and S-d-Bact-0785-a-A-21 (29 (link)) containing unique adapters for the Nextera XT indexing kit according to the manufacturer's instructions (Illumina Inc., San Diego, CA). Fungal ITS2 regions were amplified using separate adapter primers derived from IST3_KYO1 and IST4_KYO1 (30 (link)). After adding unique indices, each sample's DNA concentration was determined using the Quant-iT PicoGreen kit (Molecular Probes, Inc., Eugene, OR) and equal mass portions of each were pooled prior to sequencing.
The amplicon pools were paired-end sequenced (250 cycles) using the MiSeq platform (Illumina Inc.) at either the Interdisciplinary Center for Biotechnology Research (year 1 data set, University of Florida, Gainesville, FL) or the UCF Genomics & Bioinformatics Cluster (year 2 data set, University of Central Florida, Orlando, FL).