2.5 B1K accessions chloroplast DNA sequencing
From previous DNA shot-gun sequencing we realized that chloroplast isolation and sequencing were necessary to overcome possible confounding effects of the known transfers that occur from plastid to nuclear [nuclear plastid DNAs (NUPTs)] (Richly & Leister, 2004; Yoshidaet al. , 2014; Greiner and Fridman, personal communication). Therefore, we isolated the chloroplast DNA and performed Nanopore amplicon sequencing.
Chloroplast DNA isolation
Ten grams fresh leaf tissue were taken and grinded in pestle mortar using liquid N2. Grinded powder was transferred in small beaker mixed with 100 ml isolation buffer (50 mM Tris-HCl pH 8.0, 0.35 M sucrose, 7 mM EDTA, 5 mM 2-mercaptoethanol, 0.1% BSA), kept in 4°C for 5-30 min followed by filtration using 8 layer of gauge. The filtrate centrifuged at 1000Xg for 15 min and pellet dissolved in 3 ml of isolation buffer. The dissolved solution were loaded on top of 45/20% sucrose gradient (50 mM Tris-HCl pH 8.0, 0.3 sorbitol, 7 mM EDTA, sucrose 20% / 45%). Tubes centrifuge at 2000Xg for 30 min. Carefully middle layer (sucrose gradient junction) transferred to new tube and filed with 3 times volume of isolation buffer followed by centrifugation at 3000Xg for 20 min. Pellet (isolated chloroplast) was used for DNA isolation using CTAB method.
B1K accessions PCR barcoding and Nanopore Sequencing
We PCR amplified the region 19409-24572 using chloroplastic DNA of B1K accessions with LongAmp Taq-polymerase 2X mix (M0287S). Two-step PCR performed to tag with 96 different barcode carrying the universal adapter sequence (Table S1 ). After the barcoding all samples pooled together and purified by precipitation. 1µg of barcoded pooled PCR product was taken for library preparation using kit as per manufacturer protocol (SQK-LSK109 kit, Nanopore). 5-50 fmol DNA loaded in Nanopore flow cells (R9.4.1) after priming. After 24 hr run data were collected and performed the basecalling and demultiplexing with Guppy 5.0. Further barcodes aligned to reference with BWA-MEM2 using (-x ont2d) criteria and BAM files are sorted by coordinate. BAM files used as input for variant calling using Medaka version 1.4.4. VCF files from every alignment merged using a custom R script demultiplexing.