DNA-Seq of leaf: Dendrobium chrysotoxum
1 AbstractGenome resequencing is the sequencing of the genome of a known species and the analysis of individual or population samples. Genetic resequencing can assist researchers in identifying single nucleotide polymorphic loci (SNPs), copy number variation (CNV), insertion Variation (CNV), Insertion, Deletion and other types of variation.2 Experimental procedure2.1 Sample testingSample testing involves the concentration, integrity and purity of the sample. Concentration is determined by fluorescence quantification or enzyme markers. The integrity and purity of the samples are checked using agarose gels (agarose gel concentration: 1% voltage: 150 V, electrophoresis time: 40 min).2.2 Sample interruption1 g of genomic DNA was taken and interrupted ultrasonically using a Covaris instrument.2.3 Fragment size selectionThe beads of the interrupted sample were selected so that the sample bands were concentrated around 200-400 bp.2.4 End repair, addition of "A" and jointingThe reaction system is prepared and the reaction time is set at a moderate temperature to repair the end of the double-stranded cDNA and add an A base to the 3' end.2.5 PCR reaction and product recoveryThe PCR reaction system is prepared and the reaction program is set up to amplify the liganded product. The amplification products were recovered by purification using magnetic beads.2.6 PCR product cyclisationAfter denaturing the PCR product to single stranded, the cyclisation reaction system is prepared and mixed thoroughly for a certain period of time at the appropriate temperature to obtain a single stranded cyclic product and the final library is obtained by digesting the uncycled linear DNA molecules.2.7 Library assay The cyclisation product is tested for concentration prior to loading.2.8 On-board sequencing of qualified libraries (DNBSEQ): single-stranded circular DNA molecules are replicated by ring rolling to form a DNA nanoball (DNB) containing more than 300 copies. The resulting DNBs are then sequenced using high-density DNA nanochip technology.The resulting DNBs are added to the mesh pores of the chip and sequenced by combined probe anchored polymerisation (cPAS).Translated with www.DeepL.com/Translator (free version)