Hiseq x

Four mice from each group of day 5 (D5) Scgb1a1‐CreER^TM/+;R26R^tdTomato/+, D5 Lats1^fl/fl;Lats2^fl/fl;Scgb1a1‐CreER^TM/+;R26R^tdTomato/+, and day 14 (D14) Lats1^fl/fl;Lats2^fl/fl; Scgb1a1‐CreER^TM/+; R26R^tdTomato/+ were used. Isolated primary lung epithelial cells from the same group were pooled and sorted for DAPI^–CD31^–CD45^–EpCAM⁺tdTom⁺ cells using BD AriaII FACS sorter. The purified tdTom⁺ cells were then processed using the droplet‐based 10× Genomics Chromium approach. In brief, Gel beads in Emulsion (GEMs) were prepared using barcoded Chromium Single Cell 3’Library & Gel Bead Kit v3 (PN1000075) and Chromium B Chip Kit (PN1000154). To recover 2 × 10³ cells for control and 4 × 10³ cells each for D5 and D14 samples, approximately 5–8 × 10³ cells were loaded onto the Chromium controller. Immediately after GEMs formation, they were reverse transcribed, and the resulting cDNAs were tagged with Unique Molecular Index (UMI) and cell barcodes. Next, GEMs were broken, and cDNA was amplified and quantified using Agilent 2100 Bioanalyzer. The final 3’ Gene Expression Library was constructed by enzymatic fragmentation and size selection. The samples were then sequenced on the Illumina HiSeq X by Macrogen with a target of 2.5–3.0 × 10⁴ reads/cells (2 × 100 paired‐end reads).

Genome sequencing was performed using HiSeqX at Macrogen Inc., Seoul, Korea from extracted DNA of the WBPH sample. De novo assembly and confirmation were done by Velvet v1.2.10 [21 (link)] after filtering raw reads using Trimmomatic v0.33 [47 (link)] with default parameters. The assembled mitochondrial genome sequence was confirmed with BWA v0.7.17 [48 ] (alignment of raw reads against the assembled sequence) and SAMtools v1.9 (tview mode for a manual check of each base) [49 (link)]. All bioinformatic processes were conducted under the environment of the Genome Information System (GeIS; http://geis.infoboss.co.kr/; 24 November 2021) which have been utilized in the various genomic studies [50 (link),51 (link),52 (link),53 ].

As orthogonal sequencing data for comparison, we conducted conventional WGS on the normal sample and metastatic tumor samples. Whole genome libraries for the normal and metastatic samples were prepared and sequenced with standard TruSeq protocols. The normal and left metastatic sample were sequenced at Illumina (San Diego, CA, USA) on an Illumina 2500 with 100 by 100-bp paired-end reads, and the right metastatic sample was sequenced at Macrogen (Seoul, South Korea) on a HiSeq X with 150 by 150-bp paired-end reads. Sequence reads were aligned to the human genome version GRCh37.1 using the BWA-MEM algorithm of the Burrows-Wheeler Aligner (BWA) v0.7.4 [23 (link)] with default parameters. Read mapping and sequencing coverage statistics are listed in Additional file 1: Table S2. The GATK (v3.3) DepthOfCoverage tool was used to calculate coverage metrics [24 (link)].

Whole-genome sequencing was performed for the probands and their parents to allow compared visual examination, but data from the index case only was used for CNV calling. Libraries were prepared with the TruSeq DNA PCR Free (350) library kit and sequenced on HiSeq X (Macrogen, Korea). Raw images and base calls were generated through the integrated analysis software RTA2 (Real Time Analysis 2). Conversion of the BCL binary to FASTQ was performed with the Illumina package bcl2fastq2-v2.20.2, with demultiplexing option set to default and without trimming the adapters. Reads were aligned to the hg19 reference sequence with BWA-MEM v0.7.12q. SNVs were called with the GATK [1 (link)] HaplotypeCaller, v3.7-0-gcfedb67. Between 614,880,099 and 1,027,077,956 reads were produced per patient (average 803,031,274), with 95.46–99.8% of mapped reads. Mean coverage ranged between 27.6× and 43× (average 34×), with 40.5–88% of the reference covered at least 30× (average 67.7%). The bam file for individual NA12878 was downloaded from the Genome in a Bottle github page (https://github.com/genome-in-a-bottle/giab_data_indexes). Reference SVs sets were downloaded from the phase 3 of the 1000 Genomes Project (ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/phase3/integrated_sv_map/).

Genome sequencing was performed using HiSeqX at Macrogen Inc., Korea, from the extracted DNA of the six C. album. De novo assembly with confirmation was accomplished with Velvet v1.2.10 [40 (link)] after filtering raw reads using Trimmomatic v0.33 [41 (link)]. After obtaining the first draft of the chloroplast genome sequences, gaps were filled with GapCloser v1.12 [42 (link)], and all bases from the assembled sequences were confirmed by checking each base in the alignment (tview mode in SAMtools v1.9 [43 (link)]) against the assembled chloroplast genome generated with BWA v0.7.17 [44 ]. All these processes were conducted under the environment of the Genome Information System (GeIS; http://geis.infoboss.co.kr/; Park et al., in preparation) like other Amaranthaceae chloroplast genomes assembled [38 (link), 39 (link), 45 (link)–49 (link)].

Genome sequencing was performed using HiSeqX at Macrogen Inc., Korea, from the extracted DNA of the Korean A. thaliana de novo assembly, with confirmation accomplished with Velvet 1.2.10 [33 (link)] after filtering raw reads using Trimmomatic 0.33 [34 (link)]. After obtaining the first draft of the chloroplast genome sequences, gaps were filled with GapCloser 1.12 [35 (link)] and all bases from the assembled sequences were confirmed by checking each base in the alignment (tview mode in SAMtools 1.9 [36 (link)]) against the assembled chloroplast genome generated with BWA 0.7.17 [37 ]. All these bioinformatic processes were conducted under the environment of Genome Information System (GeIS; http://geis.infoboss.co.kr/; Park et al., in preparation).