Truseq dna pcr free ht library prep kit

The extracted genomic DNA from a laboratory-reared individual was used in constructing a high-throughput sequencing library with 500-bp insert size using the Illumina TruSeq DNA PCR-Free HT Library Prep Kit (Illumina, San Diego, CA, USA). The prepared library was sequenced on an Illumina Hiseq4000 Sequencer using the Hiseq Reagent Kit v3 (Illumina, San Diego, CA, USA) by Beijing BerryGenomics Co., Ltd. The paired-end 150 bp raw data were trimmed by removing the low quality reads using Trimmomatic 0.36 [24 (link)] and then the sequences were evaluated by FastQC v 0.11.5 [25 ]. The genome size of P. solenopsis was estimated by JELLYFISH v2.2.6 software with a K-mer method [26 (link)]. IDBA was used to assemble the generated genomic sequences with K-mer from 20 to 140 [27 ].

End-repair, A-tailing, adaptor ligation, initial, and second purification were performed using the TruSeq DNA PCR-Free HT Library Prep Kit (Illumina, San Diego, USA; [24 ]) according to the manufacturer’s recommendations [22 , 23 ], with the exception that supplied sample purification beads were replaced by AMPure XP beads (Beckman Coulter, CA, USA). To ensure balanced pooling, each library sample was quantified in duplicate by means of qPCR using the KAPA SYBR FAST Universal qPCR Kit (Roche) following [26 ] on an Applied Biosystems 7500 Fast Real-Time PCR instrument. Data analysis was performed using the HID Real-Time PCR Software v 2.3. Based on qPCR results, samples were diluted and normalized to 4 nM and equally pooled according to [22 ]. Sequencing was performed on the MiSeq FGx instrument (Verogen, [27 ]) using a 500 cycles MiSeq Reagent Kit v2 for 2 × 250 paired-end sequencing (Illumina, [28 ]) according to the manufacturer’s recommendations. The final library concentration was 12 pM with approx. 6.6% spiked PhiX control library (Illumina) following [22 ].