Nextseq 500 platform

For 15 of 16 patients, exome sequencing was performed. GL-1 to GL-13 and GL-19 exome library preparation was done with Agilent SureSelect Human All Exon V5 (Agilent Technologies, Santa Clara, CA), and sequencing was performed by Genome Diagnostics Nijmegen (https://www.genomediagnosticsnijmegen.nl) using the NextSeq 500 platform (Illumina, San Diego, CA). The ARG13 sample was enriched with the Illumina TruSeq Rapid Exome Capture Kit (Illumina) and sequenced on the NextSeq 500 platform by the Institute of Genetic Medicine at Newcastle University in Newcastle, UK. Read mapping and variant and copy number variation (CNV) calling for all samples were performed using the in-house pipeline of the Radboudumc Genome Technology Centre. Homozygosity calling was performed using RareVariantVis (Stokowy et al., 2016 (link)).

RNA samples of sufficient quality (RNA Integrity Number, RIN ≥ 7.0) and quantity were subjected to direct sequencing. Alternatively, when RNA was not deemed satisfactory, genomic DNA was sequenced. Both RNA and DNA from a single sample (T7) were sequenced.
Strand-oriented RNA-Sequencing: for each RNA sample, a directional library was prepared using the TruSeq Stranded Total RNA Sample Prep Kit (Illumina, San Diego, CA, USA), according to the manufacturer’s instructions. Ribosomal RNA depletion was performed using Illumina Ribo-Zero Epicentre kits. The cDNA libraries thus obtained, were checked for quality and quantity and finally sequenced on the Illumina NextSeq500 platform for the target production of 150 M 100-bp PE reads.
Whole genome sequencing: DNA was subjected to library preparation using the TruSeq DNA PCR-free Sample Prep kit (Illumina, San Diego, CA, USA), including inserts from 200 to 500 bp, approximately. The library was sequenced on an Illumina NextSeq500 platform on a 2 × 100 bp PE sequencing run.
In both RNA and DNA sequencing, samples were processed as indexed pools, using NextSeq 500 High output kits v2 (300 cycles).

The genetic diversity analysis of I. cangae individuals was performed using the genome skimming approach (Figure 2a) (Malé et al., 2014 (link); Weitemier et al., 2014 (link); Wessinger et al., 2018 (link)). Shotgun paired‐end libraries were constructed from 50 ng of isolated DNA. For that, samples were subjected to a random enzymatic fragmentation in which the DNA was simultaneously fragmented and bound to adapters using the QXT SureSelect kit (Agilent Technologies). The fragmented DNA was purified using AmPure XP beads (Beckman Coulter) and subjected to an amplification reaction using primers complementary to the Illumina flowcell adapters. Amplified libraries were again purified using AmPure XP beads (Beckman Coulter), quantified using the Qubit 3.0 Fluorometer (Thermo Fisher Scientific Inc.), and checked for fragments size in the 4,200 TapeStation (Agilent Technologies®) using a ScreenTape DNA 1,000 kit (Agilent Technologies). The libraries were adjusted to a 4 nM concentration, pooled, denatured, and diluted to a running concentration of 1.8 pM. The sequencing run was performed in the NextSeq 500 Illumina platform using a NextSeq 500 v2 kit high output (300 cycles).

For the RNA‐Seq analysis, the DriveMap^TM library preparation method (Selecta) was used. For cDNA synthesis, each embryo was lysed in 1× TCL lysis buffer, deposited in separate wells of a TurboCapture 96 mRNA plate (Qiagen) and incubated at room temperature for 1 hour After washing the plate three times with cold TCW washing buffer (Qiagen), 10 µL of RT reaction master mix solution was added. The plate was then subjected to incubation at 50°C for 40 minutes, followed by RT inactivation at 95°C for 5 minutes.
For the first round of gene‐specific primer (GSP) extension, 10 µL of cDNA and 10 µL of multiplex DNA polymerase master reaction mix (pool of forward GSP) were mixed and incubated for 30 minutes at 64°C. After the primer removal reaction, the plate was incubated for 30 minutes at 37°C and 5 minutes at 95°C. The second extension was conducted under the same conditions as the first extension, and the reaction was terminated by the addition of primer removal reagent mix and incubation at 37°C for 30 minutes. Using a unique combination of Fwd and Rev IND PCR primers, the first (20 cycles) and 2nd (9 cycles) rounds of PCR were performed to amplify the DNA. The amplified DNA was purified and subjected to sequencing using the NextSeq500 Illumina platform.

The sequencing was completed by Exiqon A/S Company on the NextSeq500 Illumina platform, using single‐end read and 50 nt as number of sequencing cycles.
Data preparation and analysis was based on Cap‐miRSeq pipeline (Sun et al., 2014 (link)) following these steps: (i) FASTQC and Cutadapt were applied to verify the quality of the data before and after the adapters’ trimming. The samples showed overall good data quality with the vast majority of the data obtained, presenting higher Q‐score than Q30, when a score of 30 equals an accuracy of 99.9% for the base‐calling (Cock et al., 2010 (link)); (ii) miRDeep2 was used to identify miR expression profiling where the sequencing reads were mapped to the reference human genome in miRBase v.21; (iii) normalization process was performed by Tags Per Million (TPM) method according to the total tag count in each sample. Data were filtered for count threshold, ≥20 TPM to achieve a robust validation using a different technique as RT‐qPCR; (iv) differential expression analysis was implemented by EdgeR statistical software package in R v3.6.3, applying the glmQLFTest and data were considered significant with a p value <0.01.