Nextera mate pair library prep kit

Genomic DNA extraction from M. aeruginosa cells was performed using a combination of the potassium xanthogenate-sodium dodecyl sulfate and phenol/chloroform/isoamyl alcohol procedures, as described previously (Tillett and Neilan, 2000 (link); Yoshida M. et al., 2005 (link)). The extracted DNA was sheared using a Covaris M220 focused-ultrasonicator (Covaris, Woburn, MA, United States) to an average size of 300 bp. A mate pair library was then prepared using a Nextera Mate Pair Library Prep Kit (Illumina, San Diego, CA, United States) according to the manufacturer’s instructions. The mate pair library was sequenced using a MiSeq Reagent Kit v3 (2 × 150-bp read length; Illumina) and the Illumina MiSeq platform, and assembled using SPAdes ver.3.7.0 (Bankevich et al., 2012 (link)). Open reading frames (ORFs) were predicted using GenemarkS (Besemer et al., 2001 (link)), and predicted ORFs were annotated by blastp analysis against the National Center for Biotechnology Information (NCBI) non-redundant database (nr) (E-value thresholds of < 1e^-3).

Mate-pair libraries were built for each animal carrying a CNV (Supplementary Data 6) using the Nextera mate pair library prep kit (Illumina®, Vancouver, British Columbia, Canada). Each library was sequenced using a NextSeq 500 (Illumina®) sequencer and ~20% of a high-output flow cell. Following alignment to the mm10 genome using bwa (version 0.7.12), the regions identified by aCGH were visually inspected using the Integrative Genomics Viewer^{46 (link)} to pinpoint the CNV breakpoint sequence and elucidate the mechanism for formation. The sequence data were also compared against the MutaMouse and C57BL/6J haplotype information to characterize the parental origin of each CNV. Loss of the allele in deletions or higher proportion of the allele in duplications would indicate that the CNV originated in a germ cell of the parent carrying that allele.

Sequencing libraries for the ZnDsv-02 genome were prepared using the TruSeq DNA PCR-Free Library Prep Kit and the Nextera Mate Pair Library Prep Kit (Illumina, Madison, WI, USA). Sequencing was performed using the MiSeq Reagent Kit v3 (600 cycles) on the Illumina MiSeq platform. After adapter removal and quality trimming using cutadapt [41 ] and prinseq [42 (link)], respectively, the resulting reads were assembled into contigs using SPAdes 3.10.1 [43 (link)]. The contigs and mate-pair reads were used to generate scaffolds with SCARPA 0.241 [44 (link)]. Contigs and scaffolds exhibiting the highest similarity to genome sequences of the genus Desulfovibrio or ‘Ca. Adiutrix intracellularis’ Adiu1 (LQAA00000000) by BLASTn searching against the NCBI non-redundant (nr) nucleotide database were extracted and treated as ‘trusted contigs.’ Sequence reads of 2–5 kb in length, which were obtained on a PacBio RSII platform as described previously [4 (link)], were added and re-assembled with the ‘trusted contigs’ using SPAdes 3.10.1. The resulting assemblies were manually inspected and curated as needed. After these processes, the Desulfovibrio genome was still fragmented to 55 contigs. Thus, we incorporated an assembling process as described in the Supplementary methods, and the final contig set was then obtained.

DNA extracted from diploid needles was used to build three mate-pair (MP) libraries of increasing insert size of 1,500 bp, 3,000 bp and 8,000 bp (MP1500, MP3000, MP8000). Libraries were prepared using the Nextera Mate Pair Library Prep Kit (Illumina) using the gel-plus protocol selecting for three different distribution sizes according to the manufacturer’s instructions. After fragmentation, bands of 1.5, 3 and 8 Kb were selected for circularization. The following amounts of size-selected DNA were used for the circularization reaction: 270 ng (1.5 Kb), 285 ng (3 Kb), and 97.4 ng (8 Kb).
All three MP libraries were sequenced on HiSeq2000 (Illumina) in paired-end mode with a read length of 2 × 101 bp using TruSeq SBS Kit v4. Image analysis, base calling and quality scoring of the run were processed using the manufacturer’s software Real Time Analysis (RTA 1.18.66.3) and followed by generation of FASTQ sequence files by CASAVA.

1% agarose gel electrophoresis and a Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) were used to verify DNA accuracy. The DNA library was constructed using a Truseq Nano DNA Library Kit (Illumina, San Diego, CA, USA) and a Nextera Mate Pair Library Prep Kit (Illumina) according to the manufacturer’s instructions. To produce DNA fragments of the desired size, 0.2 µg high-MW genomic DNA was randomly selected for sample library preparation in the Covaris S2 System (Covaris, LLC). Capillary electrophoresis and a Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) were used to assess the reliability of the amplified libraries. For RNA-Seq, total RNA was extracted from the flowers, buds, berries, roots, and stems of S. koreensis and the flowers, leaves, roots, and stems of S. flavescens. The RNA libraries were sequenced on a NovaSeq 6000 Platform (Illumina).

Three genomic DNA libraries were constructed according to the Illumina recommendations - two mate-pair (MP) libraries from 5 Kb and 10 Kb long fragment sizes using the Nextera Mate Pair Library Prep Kit (Illumina, USA) and one paired-end (PE) library with an insert average size of ~300 bp using the TruSeq DNA Library Prep Kit LT (Illumina, USA) according to the manufacturer’s recommendations. The whole genome sequencing was performed by the Genotek company (Moscow, Russia) on the Illumina HiSeq 2500 with 2 × 75 bp PE and 2 × 100 bp MP sequencing.