Truseq dna pcr free low throughput library prep kit

An overview of the three library construction methods used in this study is shown in Figure 1. All genomic samples for library construction were quantified using Qubit 3.0 (Invitrogen, Waltham, MA, USA). For PCR-free-frag library construction used by rCNV-seq (Method 1), gDNA (10–40 ng) was initially treated by dsDNA fragmentase at 37 °C (NEBNext dsDNA Fragmentase, New England Biolabs, Ipswich, MA, USA) to produce smaller derivatives with an average size of ~200 bps. Prepared gDNA fragments were then end-repaired, A-tailed and then ligated with barcoded sequencing adaptors using a proprietary DNA repair kit (KR2000, Berry Genomics, Beijing, China) to generate libraries for sequencing. For PCR-free-soni (Method 2) and PCR-soni (Method 3) used for commercial CNV-seq, gDNA (1 µg, PCR-free-soni; 100–200 ng PCR-soni) was sheared by sonification and fragments of 350 bps size selected on agarose gels using TruSeq DNA PCR-Free Low Throughput Library Prep Kit (Illumina, San Diego, CA, USA) and TruSeq Nano DNA Low Throughput Library Prep Kit (Illumina, San Diego, CA, USA), respectively (Figure 1).

Genomic DNA was extracted from pooled cwc15-1 seedlings 6 days after germination (6 dag), using the Qiagen DNeasy Plant Mini Kit. Libraries for DNA Next Generation Sequencing (NGS) were prepared with 1 µg DNA, using the Illumina TruSeq DNA PCR-free Low Throughput Library Prep Kit and Single Indexes Set A, and sequenced on an Illumina HiSeq 2000 machine. The transgenic insertion site in cwc15-1 was initially determined by aligning sequencing reads to the Arabidopsis genome (https://www.araport.org/data/araport11) and the border region of the transgenic construct, using CLC Genomics Workbench software version 10.1.1. The insertion site was confirmed by PCR genotyping of border regions, using transgenic and genomic primers (LB and cwc15-1 genotyping start primers, 341 bp; RB and cwc15-1 genotyping stop primers, 323 bp), and subsequently by Sanger sequencing of PCR products.

Libraries for PCR‐free short read sequencing were prepared from MN106‐Ref genomic DNA using the TruSeq DNA PCR‐Free Low Throughput Library Prep Kit (Illumina, San Diego, CA) in combination with TruSeq DNA Single Indexes Set A (Illumina, San Diego, CA) according to the manufacturer’s protocol. We prepared two libraries, with average insert sizes of 350 bp and 550 bp, respectively. Samples were sequenced to 125X depth (~66 Gb) on an Illumina HiSeq 2500 (Illumina, San Diego, CA) instrument with 125‐bp paired‐end reads.