DOI: 10.5061/dryad.8931zcrt3
title: CGG short tandem repeat genotype predictions

description: As expansions of CGG short tandem repeats (STR) are established as the
 genetic aetiology of many neurodevelopmental disorders, we aimed to
 elucidate the inheritance patterns and role of CGG STRs in autism-spectrum
 disorder (ASD). By genotyping 6,063 CGG STR loci in a large cohort of
 trios and quads with an ASD-affected proband, we determined an
 unprecedented rate of CGG repeat length deviation across a single
 generation. While the concept of repeat length being linked to deviation
 rate was solidified, we demonstrate how shorter STRs display greater
 degrees of size variation. We observed that CGG STRs did not segregate by
 Mendelian principles, with a bias against longer repeats, which appeared
 to magnify as repeat length increased. Through logistic regression, we
 identified 19 genes that displayed significantly higher rates and degrees
 of CGG STR expansion within the ASD-affected probands (p &lt; 1 x
 10-5). This study not only highlights novel repeat expansions that may
 play a role in ASD but also reinforces the hypothesis that CGG STRs are
 specifically linked to human cognition.
description: Study
 cohorts and whole genome sequencing Within
 this project, whole genome sequencing data was obtained from 5,889 samples
 from the MSSNG project and 501 from the NGC project. For the MSSNG cohort,
 PCR-free WGS was conducted on DNA samples of 1,811 trios of an
 autism-affected proband and unaffected parents and 114 quads trios of an
 autism-affected proband, an unaffected sibling, and unaffected parents.
 Library preparation and sequencing were conducted on the Illumina HiSeq X
 platform.  All samples were aligned to the GRCh38/hg38 reference genome
 using BWA-mem. Full details on the MSSNG data pipelines can be obtained
 from their website (Via MSSNG, https://research.mss.ng/). For the NGC cohort, PCR-free WGS was conducted on DNA samples obtained from blood of 167 trios of an affected proband (young children needing intensive care) and unaffected parents. DNA samples were shipped to Illumina (UK) for sequencing and were prepared with the Illumina TruSeq DNA PCR-Free Sample preparation kit (Illumina, Inc) as previously described (63,64). Samples were sequenced on the Illumina Hiseq 2500, and quality control and read alignment to the human reference genome GRCh38 was performed by Illumina as previously described. Average coverage obtained was 30–40 × for the nuclear genome and 800–1000 × for the mitochondrial genome. Genome-wide CGG STR genotyping through ExpansionHunter Genome-wide CGG repeat genotyping was conducted on the CRAM files, aligned to GRCh38, generated by the WGS described in the previous section. The short tandem repeat genotyping algorithm ExpansionHunter (version 5.0.0) was used, developed by Dolzhenko et al. The default parameters were used, and the GRCh38 FASTA file was used for the genome reference argument. For the “–variant-catalog” argument, a custom CGG-repeat catalogue JSON file was used as developed and described previously by Annear et al., however, it was updated for the GRCh38 reference assembly. The resultant output VCF and JSON files were processed through a bioinformatic pipeline, STaRparse (https://github.com/CognitiveGenetics/STaRparse), in order to automatically parse, filter, analyse, and annotate the extracted CGG STR data. STaRparse was build utilising Python (3.6.8) and R (3.6.3) environments. To ensure accuracy of repeat length predictions, loci were excluded based on sequence coverage and the presence of only flanking reads.