Neb next ffpe dna repair mix kit

Genomic DNA of the gonads from male, female, and intersex eels were extracted using the HiPure Tissue & Blood DNA Kit (Magen). The concentration and quality of DNA were detected using an Agilent 2100 Bioanalyzer (Agilent Technologies) and the integrity was detected using 1% agarose gel electrophoresis. Two microgrammes of gDNA was repaired using a NEB Next FFPE DNA Repair Mix kit (New England Biolabs). The ONT template prep kit (Oxford Nanopore Technologies) was used to prepare the template according the manufacturer’s instructions. The library was sequenced using the ONT sequencing reagent kit (Oxford Nanopore Technologies) according to the manufacturer’s instructions on the ONT PromethION48 platform with PromethION Flow Cells [20 (link)]. The fast5 format data were converted to fastq format for QC analysis using Basecall with ONT’s Guppy software [21 ]. The fastq data were further filtered to remove the adapters, short reads (length < 500 bp), and low-quality reads (MeanQual < 6).

FFPE tumor tissue samples of 78 patients consisting 20 cancer types were retrospectively collected from The First Affiliated Hospital of Zhengzhou University. DNA was extracted from FFPE tumor tissues using TIANamp Genomic DNA Kit DP340 (Tiangen, Beijing, China). DNA extracts were sheared into 200–300-bp fragments using the Picoruptor (Diagenode, Liege, Belgium). Damaged bases of fragmented DNA were repaired with the NEBNext FFPE DNA Repair Mix Kit (NEB, Ipswich, MA, USA). The extracted DNA from FFPE sections was bisulfite-converted and purified using the EZ DNA Methylation-Gold Kit (Zymo Research, Orange, CA, USA). The bisulfite-converted DNA libraries were subsequently generated with an in-house protocol. An amount of 80 ng input DNA was found to be sufficient for the preparation of targeted bisulfite sequencing libraries. And we used it for all DNA sequencing samples.
Capture probes targeting the 200 selected CpGs were individually synthesized and 5’-biotinylated by Integrated DNA Technologies (IDT, Coralville, IA, USA). Hybridization capture-based target enrichment was carried out using NadPrep Hybrid Capture Reagents Kit (Nanodigmbio, Nanjing, China). Target capture libraries were sequenced on an Illumina NovaSeq 6000 sequencing platform.

Nanopore sequencing of 2 μg of gDNA was prepared using an NEB Next FFPE DNA Repair Mix kit (New England Biolabs) and subsequently processed using the ONT Template prep kit (Oxford Nanopore Technologies). The large segment library was premixed with loading beads and then pipetted onto a previously used and washed R9 flow cell. The library was sequenced on the ONT PromethION platform with the Corresponding R9 cell and ONT sequencing reagents kit (EXP-FLP001.PRO.6; Oxford Nanopore Technologies). The genome was assembled based on three ways: initial wtdbg2 (Ruan and Li, 2020 (link)) assembly and then SMARTdenovo assembly (Liu et al., 2021 (link)), followed by error correction using racon (Vaser et al., 2017 (link)) software and adjustment by Pilon (Walker et al., 2014 (link)) software. The assembly results were evaluated by the ratio of sequencing reads, Core Eukaryotic Genes Mapping Approach (CEGMA; Parra et al., 2007 (link)), and Benchmarking Universal Single-Copy Orthologs (BUSCO; Simão et al., 2015 (link)).