Nebnext dna library prep kit

Shearing of 0.5–1 µg genomic DNA to a mean of 200–300 bp fragments was performed using the Covaris E210 focused acoustic energy system (Covaris, Woburn, MA). Whole-genome libraries were prepared using either the NEBNext DNA Library Prep kit (New England Biolabs, Ipswich, MA) or KAPA Hyper Prep kit (Kapa Biosystems, Wilmington, MA) according to the manufacturer’s protocol. Illumina compatible paired-end adapters were used, and the adapter-ligated DNA fragments were amplified by ligation-mediated PCR (KAPA Biosystems, Wilmington, MA) using a reverse PCR primer containing a six nucleotide barcode that allowed for multiple samples to be pooled and sequenced in the same run. The library was enriched for exonic sequences with the SeqCap EZ Human Exome Library v3.0 capture system (Roche NimbleGen, Madison, WI). The libraries were then sequenced with a 100 bp paired-end protocol on the Illumina HiSeq 2500 according to standard manufacturer’s protocol (Illumina, San Diego, CA).

Peripheral blood samples from the affected child and parents were obtained at the Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital for DNA extraction. DNA extraction and library preparation were performed using standard protocol and WGS was performed by Novogene Co. Ltd. (Durham, NC, USA) on HiSeq X platform.
A total amount of 1.0 μg DNA per sample was used as input material for the DNA sample preparations. Sequencing libraries were generated using NEBNext^® DNA Library Prep Kit (New England BioLabs, Ipswich, MA, USA) following the manufacturer’s recommendations and indices were added to each sample. The genomic DNA is randomly fragmented to a size of 350 bp by shearing, then DNA fragments were end-polished, A-tailed, and ligated with the NEBNext adapter for Illumina sequencing, and further PCR enriched by P5 and indexed P7 oligos. The PCR products were purified (AMPure XP system; Beckman Coulter, Indianapolis, IN, USA) and resulted libraries were analyzed for size distribution by Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and quantified using real-time PCR.

Purified genomic DNA was digested by three different restriction enzymes separately (DpnI, DpnII, and CviAII). Digested DNA segments were further sheared to ~ 250 bp by sonicator (Bioruptor, Diagenode). DNA segments were end-repaired, 3′-adenylated, and ligated to sequencing adaptor by using NEBNext® DNA Library Prep Kit (NEB, Cat. No. E6040S). After PCR amplification for 15 cycles, purified DNA library was constructed for NGS (Illumina, HiSeq 2500). Detailed protocol can be found in References [6 (link), 9 (link)].

Libraries for next-generation sequencing were prepared using the NEBNext DNA Library Prep kit (New England Biolabs) with the omission of the PCR amplification step; 100 nt paired-end sequencing was performed on an Illumina HiSeq2000 instrument (Genomic Sequencing and Analysis Facility, The University of Texas at Austin). SNP array hybridization was performed at DNA Landmarks (St-Jean-sur-Richelieu, Quebec, Canada) using a 60,000-sample chicken SNP chip developed by Illumina Inc. for the GWMAS Consortium (Groenen et al. 2011 (link)).

Genomic DNA was isolated from 30 pooled 3 dpf eis^te296f embryos using standard methods (Westerfield, 2000 ). Next-generation sequencing (NGS) library preparation was performed with the NEBNext DNA Library Prep Kit (New England BioLabs) according to the manufacturer's specifications (average insert size 250 bp). The library was sequenced on a single Illumina HiSeq 2000 lane in paired-end mode with 50 bp read length. Data were analysed using the MegaMapper pipeline as described (Obholzer et al., 2012 (link)); for further details, see the supplementary material Methods.

The extracted genomic DNA was used for library construction using NEBNext^® DNA Library Prep Kit (New England Biolabs, MA, USA), and sequencing (150 bp paired end) was conducted at Novogene Co. Ltd. (Beijing, China) using Novaseq 6000 sequencing.