Hiseq x sequencing system

Genomic DNA was extracted from cells using genomic extraction kit (Axygen, USA) and underwent WES according to the manufacturer's protocols. The exomes were captured using SeqCap EZ Exome V3 (64Mb, Nimblegen, USA) and sequenced on an Illumina HiSeq X Sequencing System (Illumina, San Diego, CA, USA) at Shoudu Technical Service Company (Suzhou, China). All single nucleotide polymorphisms (SNPs) and insertions and deletions (InDels) were identified by Genome Analysis Toolkit v.3.8 (GATK best practices).

Genomic DNA was extracted from a tissue sample of a voucher male specimen of Ancistrus triradiatus (Fig. 1), voucher number MHNG2786065 of the Natural History Museum of the City of Geneva collection. Total DNA was extracted using the PeqGold Tissue DNA Mini Kit (PeqLab). The short-read sequencing was done on a HiSeq X Sequencing System (Illumina), and performed at Macrogen Europe (Amsterdam, The Netherlands). The long-read sequencing was done using the PacBio technology (Pacific Biosciences) and performed at the Lausanne Genomic Technologies Facility. The Blue Pippin 15 KB protocol was used, with a 600 min cycles sequencing run, insert size of 30 KB and 1 smrt cell.

Whole Genome Sequencing and data analysis of AV and PKM-1 were done as described in Gupta et al. [14 (link)]. Briefly, sequencing was performed on the HiSeqX sequencing system (Illumina) (GeneWiz Inc. NJ, USA) according to the manufacturer’s protocol. A total of ~200 million reads (31.5 Gb) with Q30 > 29 Gb data was generated. The raw reads were filtered using fastp software (v0.19.5) using parameters -M 30–3 -5. The 2X 150 bp reads were mapped on S. lycopersicum cv. Heinz version SL3.0 using BWA-MEM (0.7.17) [24 (link)]. The data analysis and variant calling was performed as described in Gupta et al. [13 (link)]. The resulting vcf (variant calling format) files were annotated using the SIFT4G algorithm. The effect of base substitutions on protein function was determined by SIFT4G (SIFT score ≤ 0.05 is considered deleterious) using the SIFT4G-ITAG3.2 genome reference database generated by Gupta et al. [13 (link)].

Two technical replicate samples for each control i.e. Basal and test i.e. high altitude D3, D7, D14 and D21 were transferred to Illumina HiseqX sequencing system and loaded sequencing by synthesis (SBS) reagents as per Illumina recommendation to perform 2x150 paired end sequencing. There were approximately 45 million (150-nt length) reads. The median quality score was >30 across all the samples. The sequence reads were mapped to the Human reference genome (HG19) with STAR aligner (V.2.0). The STAR mapped reads were processed to remove PCR and optical duplicates using Picard tools and raw expression count was generated using feature Counts. The RNA sequencing data is submitted to Gene Expression Omnibus (GEO): Accession Number: GSE133702.

Total RNA of T24 cells (stably transfected with RBMX or empty vector) was extracted by TRIzol reagent. RNA-seq assays was performed by Novelbio Company (Shanghai, China). All samples were sequenced by an Illumina HiSeqX sequencing system with a paired-end 150 bp read length. RNA-seq reads were mapped to the human genome (GRCh37, primary assembly). Differential alternative splicing was quantified using the rMATS program in a HISAT2 output bam file [60 (link)]. To find distinct differential splicing events, the filter condition (FDR < 0.05, and ΔPSI ≥ 0.2) was executed. The RNA-seq data for this study have been deposited in the Gene Expression Omnibus (accession code GSE150548).

The extraction of mt DNA of F. elongatus was performed by employing a commercial Mitochondria Isolation Kit (Sigma-Aldrich, China). The agarose gel electrophoresis method and nanodrop detection were used for the integrity and purity of DNA. All the DNA samples were quantified via a QubitFluorometer (3.0). DNA samples were disrupted into fragments randomly via the ultrasonic method. End repair, A-tailing, index adapter adding, amplification, and purification were performed for library constriction according to the manufacturer's instructions (Illumina). These libraries were sent to commercial sequencing via an Illumina HiSeq X sequencing system at Personalbio in Shanghai, China.

Nine pairs of genomic DNA (from FFPE HGSOC tissues and corresponding normal myometrium as control) were extracted using a salting-out procedure. Whole-exome capture was performed by Sure Select Human All Exon 60 Mb kit (Agilent Technologies, Santa Clara, CA) according to the standard protocol. The captured template DNA fragments of the constructed libraries were sequenced through the Illumina HiSeqX sequencing system to generate 150-bp paired-end reads. The paired-end reads from whole-exome sequencing (WES) were first aligned to human reference genome (hg19) using BWA (version 0.7). PCR duplicates were then marked in the alignments using Picard tools (version 1.1). The somatic mutation calling was carried out by Var Scan (version v2.3.9). For each candidate somatic mutation site, the common variants in the 1000 genomes (MAF > 5%) and the intergenic or introgenic region variants were also excluded in the following analysis. The latent effects of the somatic SNP and insertion-deletion were annotated using ANNOVAR.