Truseq exome enrichment kit

DNA was extracted from blood cells using a FlexiGene DNA kit (Qiagen, Valencia, USA). Exome sequences were collected according to previously described procedures [15 (link)]. Briefly, genomic DNA was fragmented using a Covaris II system (Covaris, Woburn, MA, USA). Exon templates were isolated using the TruSeq Exome Enrichment Kit (Illumina, San Diego, CA, USA) and exome sequences were collected in a HiSeq2500 sequencer (paired-end 2×150) at 100x coverage. The total variants called from the exome data from this study have been deposited in DRYAD Digital Repository with accession ID: doi:10.5061/dryad.p236p.

DNA was isolated from peripheral blood with the use of standard procedure. At first, the DNA of affected individuals (II:3, II:6) was screened. We performed Sanger sequencing of the coding and neighboring intronic regions of the genes which role in the thyroid embryogenesis was documented in previous studies. Conventional sequencing of the following genes was performed: TTF1 (NKX2–1), TTF2 (FOXE1), PAX8, HHEX, SHH, TBX1, PSMA1, PSMA3, PSMD2, PSMD3. As the initial search failed in detection of causative mutations, samples were subjected to whole-exome sequencing (WES) with the use of Illumina platform (TruSeq Exome Enrichment Kit, minimal mean depth 218×). Ninety-nine percent of the reads were aligned to hg19 using BWA v0.7.12, Picard v1.130, GATK v3.4.0, SnpEff v4.1g. For pathogenicity evaluation, the following algorithms and databases were used: Sift, PolyPhen2, dbNSFP, FATHMM, MutationTaster v2, PhenIX, HPO database, and population data from GnomAD v2.1.1. For the evolutionary conservation measurement we used PhyloP and phastCons (Fig. 2).Fig. 2

A demonstration of the phylogenetic conservation of Gly727 (a) and Gln1347 (b)

Patients have signed informed consent form for genetic studies and publication of this report. Bioethical Committee of Poznan University of Medical Sciences approved the study (approval number 524/18).

Exome and transcriptome sequencing libraries were constructed by GENterprise Genomics (Mainz, Germany) using TruSeq™ DNA and RNA sample preparation kits (Illumina, Eindhoven, The Netherlands). Exome enrichment in the gDNA library was performed with the TruSeq™ Exome Enrichment Kit (Illumina, Eindhoven, The Netherlands). Average library sizes were 465 bp for exomes and 320 bp for transcriptomes. Exome (duplicates) and transcriptome (triplicates) libraries were subjected to high throughput sequencing on the Illumina HiSeq 2500 platform (NGS Unit, Johannes Gutenberg University, Mainz) generating 100 bp paired-end reads.

Genomic DNA was isolated from whole blood, using the QIAamp DNA Blood Midikit (Qiagen), according to the manufacturer’s protocol. Exonic regions of genomic DNA of three affected subjects (IDs III:5, III:8, and IV:4) were enriched using either the TruSeq™ Exome Enrichment Kit (Illumina) or the Agilent Haloplex Exome kit based on DNA digestion and capture. Exomes were barcoded and sequenced at multiple sites on the Illumina HiSeq1000 platform, and either 2 × 76-bp (TruSeq) or 2 × 100-bp (Haloplex) PE libraries, using TruSeq SBS Kit v3–HS (Illumina) reagents and a TruSeq PE Cluster kit v.3-cbot-HS flow cell. Average coverage for all the experiments was 70x and at least 20x for 89% of the target. Paired sequencing reads were aligned to the reference genome (UCSC, hg19 build) using BWA [16 (link)] and sorted with SAMtools [17 (link)] and Picard (http://broadinstitute.github.io/picard/). Post-alignment processing (local realignment around insertions-deletions and base recalibration), SNV, and small insertions-deletions (ins-del) calling were performed with Genome Analysis Toolkit (GATK) [18 (link)] with parameters adapted to the haloplex-generated sequences. The called SNV and ins-del variants produced with both platforms were annotated using ANNOVAR [19 (link)].

Samples from both CD-train and CD-test panels were sequenced using Illumina TruSeq Exome Enrichment Kit and the Illumina HiSeq2000 instrument. Reads were mapped to the human genome build hg19. Samples of each panel were called together using Genome Analysis Toolkit (GATK version 3.3-0) Haplotype Caller [22 (link)]. Variant calls were restricted to the TruSeq exome target. VCF data from WTCCC and GTEx panels were downloaded from European Genome-Phenome Archive and dbGaP, respectively. The VQSR (Variant Quality Score Recalibration) [22 (link)] method was employed to identify true polymorphisms in the samples rather than those due to sequencing, alignment, or data processing artifacts. For each VCF file, we ran ANNOVAR [23 (link)] to identify all variants mapping to Swiss-Prot proteins [24 (link)]. Specifically, we extracted the RefSeq mRNA identifiers from ANNOVAR output and mapped these to Swiss-Prot. Note that if a single variant mapped to more than one protein, all proteins were included into the affected set.

Libraries for brain biopsy controls were generated using TruSeq Exome Enrichment Kit (Illumina), according to the manufacturer's protocol, and sequenced on Illumina HiSeq, generating 150bp paired end reads. The average coverage obtained was 36×. Reads were aligned to GRCh37 using Novoalign and variants were called using GATK HaplotypeCaller. Whole exome sequencing data from the MRC brain bank samples were obtained from EGA (EGAS00001001599), mainly sequenced using the Illumina HiSeq 2000 array (ega-archive.org/).
We compared the MAF of rare variants in our cases with our in-house brain control exomes, the MRC controls, and data available on population databases, specifically gnomAD (gnomad.broadinstitute.org) and Exome Aggregation Consortium (ExAC) (exac.broadinstitute.org) (figure 2).^{15 (link)} We then further checked the estimated effect of the according rare mutation using different computation predictive programs. As functional prediction scores, we used PolyPhen-2 and Sorting Intolerant From Tolerant (SIFT). These are used to help interpret the sequence variant.