Kapa hyper prep pcr free library kit

Genomic DNA was isolated from individual carcasses of fifth instar S. frugiperda larvae after dissecting the gut tissue or from legs or heads of adults using the Pure Link Genomic DNA mini kit (Invitrogen), following manufacturer’s protocols, and then quantified using a Nanodrop spectrophotometer (Thermo Scientific). Extracted DNA was then sheared randomly to between 250 and 500 bp using a Covaris M220 focused ultrasonicator (Woburn, MA), according to manufacturer’s instructions. The fragmented DNA was then ligated with dual-indices using a KAPA Hyper prep PCR-free library kit (Roche) according to the manufacturer’s directions. The ligated fragments were quantified using quantitative PCR and a KAPA Library quantification kit (Roche), and then submitted for sequencing on an Illumina HiSeqX device running a 2 × 150 bp paired-end configuration (Admera Health, NJ). Raw paired-end sequence reads for each sample (55) are available for download in the NCBI Sequence Read Archive (SRA) under SRR12044614-SRR12044668 with associated metadata available under NCBI BioProject id PRJNA640063. The raw data was processed to remove low quality reads using the CLC Genomics Workbench v9.5.2 (Qiagen) trim function with default parameters.

Genomic DNA of the pathogen was extracted as described in the previous section. Approximately, one µg of high quality genomic DNA was sheared to a size in between 250 and 500 bp with a Covaris M220 focused ultrasonicator (Covaris, Inc., Woburn, MA, U.S.A.). After shearing, fragmented DNA was ligated with dual-indices using a KAPA Hyperprep PCR-free library kit (Roche) for Illumina according to the manufacturer’s instructions. Libraries were loaded and sequenced on an Illumina HiSEQX next-generation sequencing device running a 2 x 150 bp paired-end configuration at Admera Health LLC (Plainfield, NJ) according to the manufacturer’s directions. Raw paired-end sequence data for each sample are publicly available for download in NCBI BioProject accession number PRJNA856328.