Trueseq dna pcr free kit

The seeds of T. coccineum (cultivar: Robinson mix) were obtained commercially from Sakata Seed Co., Ltd., Japan (production number, 906435). Genomic DNA was extracted from the seeds using a DNeasy Plant Mini Kit (Qiagen), according to the manufacturer’s instructions. PE and MP libraries with three different insert sizes (3, 5, and 8 kb) of the extracted DNA were constructed using a TruSeq DNA PCR-Free kit (Illumina) and a Nextera Mate-Pair Sample Prep Kit (Illumina), respectively. The PE and MP libraries were then subjected to 151 × 2 cycles of paired-end sequencing, using NovaSeq 6000 Illumina instruments. MS libraries of the extracted DNA were constructed using a TrueSeq DNA PCR-Free kit (Illumina). The MS library was subjected to 301 × 2 cycles of paired-end sequencing using an Illumina Miseq System. PB libraries of the extracted DNA were constructed using a SMRTbell Express Template Prep Kit (PacBio). The PB libraries were subjected to sequencing using a PacBio Sequel II system.

Genome quality check and resequencing were outsourced to Macrogen Japan (Japan). Library preparation was performed using TrueSeq DNA PCR Free Kit (Illumina, USA), and sequencing was performed using NovaSeq 6000 (Illumina, USA) under 150 bp paired-end conditions to obtain sequence data in fastq format files. Sequence data were aligned and mapped to a reference genome sequence file (R64-1-1) of budding yeast S288C using BWA (0.7.17) [75 (link)]. Next, the alignment file (SAM format) was converted to a bam format file and sorted using Samtools (1.15). Variants for each sample were called and performed using Bamtools (1.15) with "mpileup"[76 (link)] with "call" and filtered using vcfutils.pl varFilter (default parameters). Variants were annotated by snpEff (4.1) [76 (link),77 (link)] using R64-1-1.86. A comparison of An and Ev variations was performed by bcftools isec. Called variants were checked manually using IGV (2.8.10) and validated by chi-square test for base composition between An and Ev.
The threshold for validation was set at an FDR of 0.05 or less, corrected by the Benjamini-Hochberg method[72 (link)]. The raw data were available in the DNA Data Bank of Japan (accession number: DRA014470).

The first batch of samples included 20 randomly selected ginseng plantlets regenerated from 12-y-old ginseng calli, and which were arbitrarily divided into two pools. The second patch of the sample included seven regenerated plantlets from 13-y-old ginseng calli. DNA extraction of the three pooled samples were conducted using a modified CTAB method [27 ]. The high-quality genomic DNA of each sample was utilized for subsequent whole-genome resequencing. Short pair-end (150 bp) DNA libraries of the three samples were constructed with Illumina Trueseq DNA PCR-Free kit at Novogene (Tianjin, China) and sequenced with the Illumina Hi-seq 4000 platform (Illumina, CA, US). The data were deposited in the SRA database of GenBank (http://www.ncbi.nlm.nih.gov/sra, accessed on 20 November 2021) with the BioProject accession number PRJNA782439. Only clean reads (base quality > 30) were retained for subsequent data analyses.