D with ratio of 1.0 fluorometer utilizing the Qubit dsDNA BRquantity was measured with a Qubit 1.0 fluorometer usingwas Qubitof DNA ing. DNA assay kit. The quantity of DNA required for sequencing the 1 dsDNA in 48 volume. quantity of this Qubit is recommended because it delivers accurate BR assay kit. The The use of DNA necessary for sequencing was 1 of DNA in 48 measurementsuse of this Qubit is recommended because it solute [25]. volume. The that will distinguish DNA from the rest from the provides correct measure-ments that can distinguish DNA in the rest on the solute [25]. 2.3. DNA Library Preparation and SequencingA DNA library was prepared in accordance using the nanopore protocol using Native 2.three. DNA Library Preparation and Sequencing Barcoding GSK854 Protocol Genomic DNA with barcode kit 1-12 (EXPNBD104) and ligation sequencing kit A DNA library was prepared in accordance with the nanopore protocol applying Native (SQK-LSK109), version NBE_9065_v109_revJ_23May2018. The DNA library preparation Barcoding Genomic DNA with barcode kit 1-12 (EXPNBD104) and ligation sequencing kit consists of quite a few measures: (i) DNA repair (FFPE) and end-prep for optimizing DNA excellent, (SQK-LSK109), version NBE_9065_v109_revJ_23May2018. The DNA library preparation (ii) preparing DNA sequence ends for barcode and adapter attachment, and (iii) preparing consists of quite a few actions: (i) DNA repair the flow cell). DNA for optimizing DNA high-quality, R9.4 flow cell for sequencing (priming (FFPE) and end-prep clean-up was performed in (ii) preparing DNA preparation step barcode and adapter attachment, The DNA library AL-8810 web involving every librarysequence ends forusing magnetic AMPure XP beads.and (iii) preparing R9.4 flow into the R9.4 MinION Flowcell, and sequencing was performed using MinKnow was loadedcell for sequencing (priming the flow cell). DNA clean-up was performed in among every library application from ONT. preparation step employing magnetic AMPure XP beads. The DNA library was loaded in to the R9.four MinION Flowcell, and sequencing was performed working with MinKnow application 2.4. Information Evaluation from ONT. two.4.1. Sequence Raw Data Evaluation The output of MinION sequencing was raw Fast5 information, which was subsequently base referred to as into FASTQ files employing the Guppy program v4.2.38aca2af8 [26,27]. Then, a information good quality check was performed employing the NanoStat plan v1.five.0 [28] to acquire the statistic of the FASTQ reads and its distribution of high quality scores. The NanoPlot system v1.33.1 [29] was employed to create a plot of reading length x average top quality score. Reads with inadequate good quality (Q 7) and length 500 bp had been filtered applying the NanoFilt plan v2.7.1 [30], and the parameters applied had been -l 500 -q 7 eadcrop 10 ailcrop ten eadtype 1D. The QC-passed reads had been assembled making use of Rebaler (v0.two.0) [31], with Dipterocarpus turbinatus (NCBI accession code NC_046842.1) because the reference-based assembly [32], for correction of reads and assembled reads into contigs. The resulting contigs had been topic toForests 2021, 12,4 ofassembly polishing using the MEDAKA consensus v1.two.1 [33] to receive contigs with high accuracy. The statistics on the polished contig were calculated making use of QUAST v5.0.2 [34] with reference to the D. turbinatus chloroplast genome. Subsequently, the polished contig was annotated working with the GeSeq platform [35], and GenBank annotations had been generated. two.4.two. Chloroplast Marker Evaluation The GenBank annotation obtained from GeSeq was visualized using SnapGene v5.two.3 [36] to search for the potential gen.