Agilent 2100 bioanalyzer on chip electrophoresis

Twenty ng of DNA were used for multiplex PCR amplification using the Ion AmpliSeq Cancer Panel (Life Technologies) that explores selected regions of the following 46 cancer-associated genes: ABL1, ALK, AKT1, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR/VEGFR2, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RET, RB1, SMAD4, SMARCB1, SMO, SRC, STK11, TP53, VHL.
The quality of the obtained library was evaluated by the Agilent 2100 Bioanalyzer on-chip electrophoresis (Agilent Technologies). Emulsion PCR was performed either manually or with the OneTouch DL system (Life Technologies). Sequencing was run on the Ion Torrent Personal Genome Machine (PGM, Life Technologies) loaded with a 316 chip as per manufacturer's protocol. Data analysis, including alignment to the hg19 human reference genome as well as variant calling and filtering, was done using the Torrent Suite Software v.3.6 (Life Technologies) with default options for the Ion AmpliSeq Cancer Panel. Variants were annotated using the SnpEff software v.3.4 [12] (link) and the NCBI mRNA Reference Sequences listed in Table S2. Alignments were visually verified with the Integrative Genomics Viewer; IGV v.2.3 [13] (link).

Total RNA was extracted from young leaves of the same individual N. yunnanensis tree using a CTAB-pBIOZOL method [17 (link)]. The purity, concentration, and integrity of RNA samples were measured on a NanoDrop One UV-Vis spectrophotometer (Thermo Fisher Scientific, USA), a Qubit 3.0 Fluorometer (Thermo Fisher Scientific, USA) and with Agilent 2100 Bioanalyzer on-chip electrophoresis (Agilent Technologies, Inc; Santa Clara, CA) [18 (link)], respectively, to ensure that the samples qualified for transcriptome sequencing. The cDNA library was prepared using the TruSeq RNA Sample Preparation Kit v2 (Illumina, San Diego, CA, USA), and then sequenced on a DNBSEQ-G50 platform at BGI-Shenzhen (BGI Co. Ltd., Shenzhen, China), resulting in ∼8.85 Gb of raw transcriptome data (150 bp, paired-end). All of the raw sequence reads were further filtered using SOAPfilter v2.2 (SOAP, RRID:SCR_000689) with the parameters “-y -q 33 -i 200 -g 1 -M 2 -Q 20” to remove the adapters and low-quality reads.

Amplicons. Deep sequencing was performed using the Ion Torrent platform (Life Technologies). Briefly, 10 ng of purified genomic DNA were used for library construction with the Ion AmpliSeq Colon and Lung Cancer Panel v2 (Life Technologies) that targets 504 mutational hotspot regions of the following 22 cancer-associated genes, in alphabetical order: AKT1, ALK, BRAF, CTNNB1, DDR2, EGFR, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FGFR3, KRAS, MAP2K1, MET, NOTCH1, NRAS, PIK3CA, PTEN, SMAD4, STK11, TP53. Emulsion PCR was performed either manually or with the OneTouch DL system (Life Technologies). The quality of the obtained library was evaluated by the Agilent® 2100 Bioanalyzer on-chip electrophoresis (Agilent Technologies; Santa Clara, CA). Sequencing was run on the Ion Torrent Personal Genome Machine™ (PGM, Life Technologies) loaded with a 316 chip as per manufacturer's protocol. Data analysis, including alignment to the hg19 human reference genome and variant calling, was done using the Torrent Suite Software v.3.2 (Life Technologies). Filtered variants were annotated using both the Ion Reporter software v1.2 (Life Technologies) and the SnpEff software v.3.0 (alignments visually verified with the Integrative Genomics Viewer; IGV v.2.1, Broad Institute) [46 (link)].