Truseq dna pcr free lt sample preparation kit

Total extracted DNA was used for high throughput sequencing (MiSeq platform, Illumina, San Diego, CA, USA) of the bacterial 16S rRNA gene or fungal ITS1 region. We amplified the V3–V4 region of 16S rRNA gene using degenerate barcoded primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′), and the ITS1 region using degenerate fungal primers ITS1-5.8Sfw (5′-CTGTAAAAGTCGTAACAAGGTTTC-3′) and ITS1-5.8Srv (5′-AAGTTCAAAGAYTCGATGATTCAC-3′). PCR amplifications (KAPA 2G Robust Hot Start DNA Polymerase, Kapa Biosystems, Hoffmann-La Roche, Switzerland) were carried out with 25 or 27 cycles, respectively. PCR products were purified and normalized with the SequalPrep™ Normalization Plate Kit (Thermo Fisher Scientific, Waltham, MA, USA). Triplicates of the amplicons were pooled, gel eluted (NucleoSpin gel clean-up, Macherey-Nagel, Düren, Germany), and ligated with sequencing adapters (TruSeq DNA PCR-free LT Sample Preparation Kit, Illumina; KAPA HyperPlus kit, Kapa Biosystems, Hoffmann-La Roche, Switzerland). Amplicon libraries were pooled in equimolar concentrations, validated by the KAPA Library Quantification Kit (Illumina, San Diego, CA, USA), and sequenced on the MiSeq platform using the 2 × 300 bp kit at the CEITEC Genomics Core Facility (CEITEC, Masaryk University, Brno, Czech Republic).

Amplicons of the 20G tDNA, 100G tDNA, and 100G adapter primer libraries, prepared as described above, were sequenced using the Illumina technique [24 (link)]. The concentration of amplicons was determined using the Agilent Bioanalyzer 2100 and DNA 7500 kit. Library preparation was performed using Illumina's TruSeq® DNA PCR-Free LT Sample Preparation Kit following the manufacturer's description, except that amplicons were not fragmented before library preparation. 100 ng of amplicon DNA were used as input material, and the protocol was started at the step of end-repair. Libraries were individually indexed as part of the protocol and quantified/qualified using the Agilent Bioanalyzer 2100 as described above. Samples were sequenced on Illumina's MiSeq in 300 bp paired-end mode using sequencing chemistry MiSeq® Reagent Kit v3 (600 cycle). The reads were extracted in FastQ format by the MiSeq control software (MCS v2.4.1.3) or bcl2fastq v2.16/v2.17.1 software (Illumina). The raw data (fastq files) of Illumina amplicon sequencing are available at the NCBI Short Read Archive (SRA) (http://www.ncbi.nlm.nih.gov/sra/) under accession number SRP098685.

Genomic DNA from whole coffee berry borers was sheared to 350 and 550 bp sized fragments. To remove any amplification bias at the PCR step of the library preparation, noPCR libraries were prepared from coffee berry borer DNA using Illumina TruSeq DNA PCR-Free LT Sample Preparation Kit according to manufacturer’s instructions. Briefly, 1–2 ug of genomic DNA was fragmented using Covaris shearing with prescribed settings for 350 or 550 bp insert sizes. The fragmented DNA was cleaned, end-repaired, and selected for appropriate insert sizes (350 bp and 550 bp) using different ratios of Sample Purification Beads. This was followed by 3′ end adenylation and adapter ligation. Libraries were sequenced in Illumina Hiseq2000 with 100 bp paired-end reads.