Cbot station

The patient's and her parents' genomic DNA were extracted from 2-ml peripheral blood samples using the QIAamp DNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany). The DNA concentration and purity were measured using an Invitrogen Qubit dsDNA detection kit and a Qubit4 fluorometer (Carlsbad, CA, USA). The Agilent Sure Select Target Enrichment System (Agilent Technologies Inc., Santa Clara, CA, US) was used to produce an adapter-ligated library according to the manufacturer's instructions. An XT Inherited Disease Panel (cat No. 5190–7519, Agilent Technologies Inc.) containing 2,742 genes was used to create the capture library. The clusters were then produced using an Illumina cBot Station and sequenced on an Illumina HiSeq 2,500 System (Illumina Inc., San Diego, CA, US). Using human genome hg19 as the reference, alignment of sequence and repeated labeling were performed using BWM version 0.7.17 (http://bio-bwa.sourceforge.net/) and Picard bioinformatics software version 2.5.0 (https://broadinstitute.github.io/picard/) for biological analysis and interpretation. GATK 4.0.0.0 (https://gatk.broadinstitute.org/hc/en-us/sections/360007407851-4-0-0-0) and Samtools 1.8 (https://sourceforge.net/projects/samtools/files/samtools/1.8/) were used to identify mutation sites.

Considering that the twins were monochorionic identical twins and twin A presented with situs inversus, WES was first performed on twin A and his parents. Twin A’s DNA sample, obtained through amniocentesis and parents’ peripheral blood DNA were used for whole-exome sequencing to identify causal genetic variants. Briefly, 3 μg DNA was sheared to fragments of 150–200 bps in size. An adaptor-ligated library was prepared using the paired-end sequencing library prep kit (Agilent Technologies, Santa Clara, CA, United States). Both coding exons and flanking intronic regions were enriched using the Agilent SureSelect XT Human All Exon V6 reagent kit (Agilent Technologies, Santa Clara, CA, United States). Clusters were then generated by isothermal bridge amplification using the Illumina cBot station, and sequencing was performed using the Illumina HiSeq 2500 System (Illumina, San Diego, CA, United States). Burrows-Wheeler Alignment tool (BWA) v0.2.10 was used for sequence alignment to the Human Reference Genome (NCBI build 37, hg 19). Data quality was assessed using FastQC (version 0.11.2). The read data were uploaded to the Ingenuity Variant Analysis platform (Qiagen, United States) for mutation screening and interpretation. Copy number detection and visualization of WES were performed using the bioinformatics tool CNVkit.2.3.

The samples were normalised and pooled for the automated cluster preparation with an Illumina cBot station (Illumina, San Diego, CA, USA). CD4⁺ T cell libraries were run on eight lanes, 8–9 samples per lane. The CD8⁺ T cell libraries and CD4⁻CD8⁻ cell fraction libraries were allocated to four pools, including 18–20 samples per pool. Each pool was run on three lanes. The samples were sequenced with an Illumina HiSeq 2500 instrument (Illumina) and TruSeq v3 sequencing chemistry (Illumina). Paired-end sequencing with 2 × 100-bp read length was used with a 6-bp index run. Technical quality of the HiSeq 2500 run was good, and the cluster amount was as expected. More than 76% of all bases above Illumina quality score Q30 was required. The sequencing runs of 37 samples were repeated due to low raw read counts (less than 30 million reads per sample). The median yield of the other samples was 47 million reads (read 1 + read 2).