Sequence control software

One hundred ng of the rRNA-depleted RNA was used for library construction using NEBNext^® Ultra Directional RNA Library Prep Kit for Illumina^® and NEBNext^® Multiplex Oligos for Illumina^® (New England Biolabs) according to the manufacturer’s instructions. The samples were analyzed using a Genome Analyzer IIx (Illumina^®). Data were processed using the Sequence Control Software (Illumina^®). Single-ended sequence reads were scanned 3′ to 5′. Those with a Phred quality value below 20 were trimmed using the FASTQ Quality Trimmer tool of the FASTX-toolkit (http://hannonlab.cshl.edu/fastx_toolkit/index.html). Reads of less than 35 bases after trimming were discarded from further analysis. Trimmed reads were aligned to the RHA1 genome40 (link) using Bowtie 2 in end-to-end mode with the “very sensitive” option41 (link). Reads of rDNA sequences were omitted in subsequent analyses. A custom Perl script was developed to create a GTF file using the recently updated RHA1 annotation by the NCBI Prokaryotic Genome Annotation Pipeline. Mapped read counts per gene were obtained using the HTSeq package42 (link). Differential expression (>2-fold change with a false discovery rate-adjusted p value < 0.05) was analyzed using the DESeq2 package43 (link). The RNA-seq data set was deposited in Gene Expression Omnibus44 (link) under accession number of GSE77158.

The raw data produced on the MGI-2000 platform were filtered and aligned against the human reference genome (hg19) using the Burrows–Wheeler Alignment tool (Li and Durbin, 2009 (link)) after evaluation according to Illumina Sequence Control Software (SCS). The single-nucleotide polymorphisms (SNPs) were called by using Genome Analysis ToolKit software (Van der Auwera et al., 2013 (link)).
Variants were annotated using ANNOVAR (Wang et al., 2010 (link)). The effects of single-nucleotide variants (SNVs) were predicted by the SIFT, Polyphen-2, and MutationTaster programs. Variants were filtered by a minor allele frequency (MAF) of < 0.1% in the gnomAD (Karczewski et al., 2020 (link)), 1000 Genome (Genomes Project et al., 2015 (link)), ExAC (Lek et al., 2016 (link)) databases and the Exome Variant Server (EVS; NHLBI Exome Sequencing Project).
All variants were interpreted according to ACMG/AMP standards and categorized as pathogenic, likely pathogenic, variants of unknown clinical significance (VUS), likely benign and benign (Richards et al., 2015 (link)). Variant validation was performed using Sanger sequencing (ABI 3730xl Genetic Analyzer).

Array capture was performed via the Agilent SureSelectXT2 Target Enrichment System as previously described [14 (link)]. Briefly, array hybridization was captured by mixing the pooled libraries with a buffer solution and oligo-blockers, which was incubated for 24 h at 65 °C. The hybridized library molecules were performed with Dynabeads® MyOne™ Streptavidin T1 (Invitrogen, #65601). The captured library was amplified as following: 21 μl 2× KAPA HiFi HotStart ReadyMix, 1 μl 5 μM primer, 20 μl captured library beads suspension. PCR amplification program was 98 °C 2 min; 98 °C 30 s; 65 °C 30 s; 72 °C 30 s, 13 cycles and a final step at 72 °C for 4 min. Purifications between procedures were conducted using Agencourt AMPure XP beads and the libraries were evaluated with Qubit dsDNA HS Assay kit (Invitrogen, Q32851). Finally, DNA libraries of the proband were analyzed by whole exome sequencing (WES). WES was carried out on the HiSeq2500 platform as paired-end 200-bp reads. Illumina Sequence Control Software (SCS) was used to evaluate the sequencing data, thus removing adapter sequences in the raw data and discarding low-quality sequencing reads. Conventional Sanger sequencing of the SPAST gene was further performed in 15 individuals from the Chinese family.