Hiseq 1500

Venous blood samples were collected from 18 children and from their parents and siblings (if present). Genomic DNA was extracted using a standard protocol.
R-WES was defined as a process completed within 5–14 days of the sample collection and included transport to the laboratory, DNA isolation, sequencing, and the first analysis of the WES results. WES was performed on the proband DNA using SureSelect Human All Exon v5 (16 patients) or v7 (two patients) (Agilent Technologies, Palo Alto, CA, USA) according to the manufacturer’s instructions. The libraries were paired-end sequenced (2 × 100 bp) on the HiSeq 1500 (Illumina, San Diego, CA, USA) in Rapid Run mode and analyzed as previously described [13 (link)].
The variants considered as disease causing were validated in proband and studied in all the available family members by direct Sanger sequencing using the BigDye Terminator v3.1 Kit (Applied Biosystems, Foster City, CA, USA) on the ABI 3500Xl Genetic Analyzer (Applied Biosystems), or by amplicon deep sequencing (ADS) performed using the Nextera XT Kit (Illumina) and sequenced on the HiSeq 1500 (Illumina).

RNA quality was assessed using the Experion RNA StdSens Analysis Kit. RNA-seq libraries were prepared from total RNA using the TruSeq Stranded mRNA LT kit according to the manufacturer's instructions. Quality of sequencing libraries was controlled on a Bioanalyzer 2100 using the Agilent High Sensitivity DNA Kit. Pooled sequencing libraries were quantified with digital PCR (QuantStudio 3D) and sequenced on the HiSeq 1500 Illumina platform in Rapid-Run mode with 50 base single reads. RNAseq was performed from RNA isolated using the RNAeasy mini kit with the Illumina Truseq mRNA kit v2 on an Illumina Hiseq 1500 according to the manufacturer's instructions.

Genomic DNA was isolated with the Qiagen Blood and Tissue Kit according to the manufacturer’s instructions. 0.5% (w/w) of unmethylated lambda phage DNA (Promega) was added to the sample genomic DNA for the purpose of an unmethylated control to measure the bisulfite non-conversion frequency in each sample. Genomic DNA was fragmented with either either a Covaris S2 sonicator or a Covaris M220 sonicator to a mean length of 200 bp, then end-repaired, A-tailed, ligated to methylated Nextflex Bisulfite-Seq barcodes (Perkin Elmer) using the NxSeq AmpFREE low DNA library kit (Gene Target Solutions) and subjected to PCR amplification with KAPA HiFi Uracil+ DNA polymerase (KAPA Biosystems)^{56 (link)}. Sequencing was performed single-end on a HiSeq 1500, NextSeq 500, or paired-end on a NovaSeq 6000 (Illumina).

A polyA fraction of the total RNA was conducted with RNA-Seq analysis using an Illumina HiSeq 1500 instrument, as previously described [27 (link)]. At least 10 million reads (1/50 nt) were generated. The RNA-Seq analysis was performed with the participation of ZAO Genoanalytika, Moscow, Russia.

Approximately 1 µg of the extracted DNA was sequenced using Pair-end Illumina HiSeq. 1500 platform at Health in Code (A Coruña, Spain). A total of 387,524,268 reads with a read size of 100 bp were generated.

Samples for RNA-seq analysis (n = 3) were prepared as described in the TruSeq^® RNA Sample Preparation Guide (Illumina). High-performance, paired-end (2 × 100 bp) sequencing was performed on an Illumina Hiseq 1500 apparatus by the Institute of Agrobiotechnology of Rosario (Rosario, Argentina). Low-quality RNA-Seq reads (QScore < Q30) detected using FastQC (Version 0.11.2) were discarded (Andrews 2010 ). De novo assembly was performed by merging the high-quality reads using Trinity software (Grabherr et al. 2011 (link)) with a minimum contig length of 200 bases and a k-mer size of 25 bp. Functional annotation of assembled transcripts was performed by homology search (BLAST/Uniprot and SwissProt), protein domain identification (HMMER/PFAM), protein signal peptide and transmembrane domain prediction (signalP/tmHMM), and annotation databases search (eggnog/GO/Kegg) using Trinotate pipeline (Bryant et al. 2017 (link)).
The reads' FPKM (Fragments Per Kilobase Million) value was determined by the eXpress abundance estimation method (Roberts and Pachter 2013 (link)). Fold change for selected transcripts was estimated by FPKM of L. corniculatus / FPKM of L. tenuis.