Truseq nano dna library preparation kit

The PowerLyzer PowerSoil DNA isolation kit (Mo Bio) was used to extract DNA from Sterivex filters according to the modified protocol of Jacobs et al. (88 (link)). Bead beating was performed singularly in a PowerLyzer 24 Bench Top Bead-Based Homogenizer (Mo Bio) at 2,000 rpm for 5 min.
DNA concentration and quality were evaluated with LabChip GX (PerkinElmer). Libraries were prepared with the TruSeq Nano DNA Library Preparation kit and sequenced in a NextSeq 500 (Illumina) with a 2 × 150-bp high-output run. Output consisted of paired-end reads with a median insert size of ∼480 nucleotides (nt).

A genomic DNA (gDNA) library was constructed for sequencing following protocols of the TruSeq Nano DNA Library Preparation Kit (Illumina, San Diego, CA). Adaptors were ligated to library fragments sheared by Covaris (Covaris, Woburn, MA, USA) and were then subjected to PCR amplification. The quantitation and abundance determination of PCR amplicons were performed on Qubit 3.0 Fluorometer (Life Technologies, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, USA), respectively. WGS was performed on HiSeq X (Illumina, San Diego, CA), with the use of Illumina bcl2fastq software version 2.15 for base calling analysis.

TA100 genomic DNA samples were sheared to fragments with a peak size of 350 bp using a sonicator (Covaris, Woburn, MA, USA). The obtained DNA fragments were used for library construction using the TruSeq nano DNA library preparation kit (TruSeq; Illumina, San Diego, CA, USA), with a slight modification for Hawk-Seq™. Briefly, DNA fragments were subjected to end repair, 3′ dA-tailing and ligation to TruSeq-indexed adaptors according to the manufacturer’s instructions. Thereafter, the DNA concentration of each sample was measured using Agilent 4200 TapeStation (Agilent Technologies, Santa Clara, CA, USA). Ligated products were diluted with suspension buffer, and 78 amol of the ligated products were PCR amplified (18 (link)). The amplified PCR products were sequenced with 2 × 100 bp or 2 × 150 bp to yield ca. 50 M read pairs using HiSeq2500 or HiSeqX (Illumina, San Diego, CA, USA).

WGS of patient samples was performed on DNA extracted from fresh frozen tissue and matched blood. 200 ng of DNA was fragmented to approximately 550 bp using a focused-ultrasonicator (Covaris M220). Libraries were prepared with the Illumina TruSeq nano DNA library preparation kit. The libraries were molecularly barcoded with IDT for Illumina TruSeq DNA Unique Dual Index adapters prior to pooling and sequencing to a depth of 40 × for the normal and 80 × or 100 × for tumour using paired 150 bp reads on the Illumina NovaSeq 6000 platform.
WGS of PDX tumours was performed on DNA extracted from fresh frozen tissue. Libraries were prepared using the Nextera Flex library method (Illumina). Indexed libraries were sequenced to a depth of 60 × using paired 150 bp reads on the Illumina NovaSeq 6000 platform.

The leaf tissue from 3-week-old plants was collected and flash-frozen in liquid nitrogen. Genomic DNA was extracted from the leaf tissue using DNeasy Plant Maxi kit (Qiagen). For the Illumina PCR-free library, genomic DNA was fragmented in a Covaris S220 and separated on a SAGE-ELF (Sage Science) following the manufacturer’s instructions. The fraction that is 310~450 bp in size from SAGE-ELF were used for PCR-free library construction using TruSeq Nano DNA Library Preparation Kit (Illumina). The 20-kb PacBio library were prepared and sequenced on PacBio RS II using P6-C4 chemistry at Tianjin Biochip Corporation, following the manufacturer’s standard protocols.
Hi-C was performed following a published protocol^{48 (link)}. Briefly, 2 g of 10-day-old broomcorn millet seedlings were fixed in 1% formaldehyde solution. The nuclei/chromatin was extracted from the fixed tissue and digested with HindIII (New England Biolabs). The overhangs resulting from HindIII digestion were filled in by biotin-14-dCTP (Invitrogen) and the Klenow enzyme (NEB). After dilution and re-ligation with T4 DNA ligase (NEB), genomic DNA was extracted and sheared to a size of 300−500 bp with Bioruptor (Diagenode). The biotin-labeled DNA fragments were enriched using streptavidin beads (Invitrogen) and subject to library preparation.

Next generation sequencing (NGS) libraries were prepared using the Illumina TruSeq Nano DNA Library Preparation Kit following standard procedures recommended by the manufacturer. Completed libraries were evaluated using a combination of Qubit dsDNA HS, Caliper LabChipGX HS DNA and Kapa Illumina Library Quantification qPCR assays. Libraries were combined in a single pool for multiplexed sequencing and this pool was loaded on one standard MiSeq flow cell (v2) and sequencing was performed in a 2 × 250 bp paired end format using a v2, 500 cycle reagent cartridge. Base calling was done by Illumina Real Time Analysis (RTA) v1.18.54 and output of RTA was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v1.8.4.
De novo assembly was performed using SPAdes (version 3.9.0) (Bankevich et al., 2012 (link)). The reads were assembled using SPAdes (version 3.9.0) and further edited by using DNASTAR (v1.12) (Nurk et al., 2013 ). Initial prediction and annotation of open reading frames (ORF) and tRNA/rRNA gene prediction were carried out with the Rapid Annotation using Subsystem Technology server (RAST) (Overbeek et al., 2014 (link)). Gene annotation was carried out by NCBI Prokaryotic Genome Automatic Annotation Pipeline (PGAAP 3.3) (http://www.ncbi.nlm.nih.gov/genome/annotation_prok/).