Magnetic beads

RNA sequencing was performed at the Johns Hopkins University School of Medicine Transcriptomics and Deep Sequencing Core, guided by the direction of Dr. Haiping Hao. RNA‐seq library for Illumina platform sequencing was prepared using Illumina TruSeq stranded total RNA Sample kit following manufacturer's recommended procedure. Briefly, 100 ng of total RNA was first depleted of ribosomal RNA with Ribozero Gold magnetic beads and further purified with Agencourt AMPure XP beads. RNA was fragmented at 94 °C for 8 min and primed with random primer. The fragmented RNA was then converted to double strand cDNA, end repaired, A tailed, and ligated with Unique Dual Indexed adaptors. The adaptor added cDNA library was then PCR amplified using the following conditions: 94 °C for 30 s, 15 cycles of 98 °C for 10 s, 60 °C for 30 s, 72 °C for 30 s, and 72 °C for 5 min. The PCR amplified library was purified using Agencourt AMPure XP magnetic beads and run out on Agilent High Sensitivity DNA Chip for quality check. Individual library was then further quantified using KAPA library quantification qPCR kit and pooled. The pooled library was sequenced on Illumina NovaSeq S1 200cycle kit for 2X100bp sequencing.

In total, 5 regions were selected for validation, which covered 18 CpGs interrogated by the 450 k array. Primers were designed for bisulfite-converted DNA using MethPrimer [37 ]. In total, 10 ng of bisulfite-converted FFPE extracted DNA from four trios was used to amplify the specific genomic regions. The hot-start enzyme KAPA HiFi Uracil + (KAPA Biosystems Inc, Wilmington, MA, USA) was used for PCR and products were cleaned using magnetic beads (Beckman Coulter Inc, Brea, CA, USA) and quantified using Picogreen reagents. Samples were tagged and pooled prior to sequencing on the Illumina MiSeq according to the manufacturer’s instructions.
Raw MiSeq paired-end reads were mapped to human genome build hg19 with Bismark v0.9.0 [38 ] using Bowtie 2 [39 ] as the aligner. Methylated and unmethylated base counts were generated with the bismark_methylation_extractor utility and exported as BedGraph files for further analysis and display in Integrative Genomics Viewer [40 ]. Aligned BAM files were sorted and indexed with SAMtools [41 ] for assessment of the regions of interest in Integrative Genomics Viewer. The number of C reads (methylated prior to conversion) was divided by the total number of reads per bisulfite-sequenced CpG site to discern the percentage methylation. These were then compared with the respective 450 k β-values to compare platforms.

A total of 600 ng of DNA (used as templates) was amplified after 25 μL of 2×Qiagen Multiplex PCR Master Mix, 5 μL of 5×Q solution, 1 μL of forward primer set pool, and 1 μL of reverse primer set pool were added to form a reaction system by using a Multiplex PCR Kit (Qiagen, Germany). Then, PCR was performed at 1 cycle of 95°C for 15 min, 10 cycles of denaturation at 94°C for 30 s, and 15 cycles of both annealing at 60°C for 90 s and extension for 30 s at 72°C. After a final extension for 5 min at 72°C, the system was cooled to 4°C. The target fragment of multiplex PCR products was purified on magnetic beads (Agencourt No. A63882, Beckman, Beverly, MA, USA).

For Illumina library construction, DNA was sheared by ultrasonication (Covaris M220 series, Woburn, MA), and the fragments end-paired, A-tailed (Lucigen NxSeq DNA prep kit, Middleton, WI), and ligated to TruSeq adapters (IDT, Coralville, Iowa); small fragments were removed twice using magnetic beads (Beckman Coulter, Danvers, MA) (White III et al., 2013a (link),b (link); White III and Suttle, 2013 (link)). No PCR enrichment was used to amplify libraries to avoid PCR duplication bias. Libraries were checked for size and adapter-dimers using a Bioanalyzer HighSens DNAchip (Agilent). Libraries were quantified using Qubit (Invitrogen, Carlsbad, CA), according to the manufacturer's instructions, by qPCR using a microfluidic digital PCR quantified standard curve (White III et al., 2009 (link)). The resulting libraries were pooled, and sequenced using both 250 and 100 bp paired-end sequencing on the MiSeq (GenoSeq UCLA Los Angeles, CA) and HiSeq (McGill University/Génome Québec, Montreal, QC) platforms, respectively.

The amplification of the V3-V4 variable region of the bacterial 16S rRNA gene via PCR was carried out using a two-step method and two primers (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACG-GGNGGCWGCAG (forward) and 5′GTCTCGTGGGCTCGGAGATGTGTATAAGAGACA-GGACTACHVGGGTATC-TAATCC-3′ (reverse)). The resulting PCR products were subjected to 2% agarose gel electrophoresis and the 16S rRNA libraries were purified using magnetic beads (AMPure XP), according to the manufacturer’s instructions (Beckman Coulter, Wycombe, UK). The sample purity was confirmed using a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). For second-round PCR, Illumina Nextera barcodes (Illumina, Inc., San Diego, CA, USA) were attached to first-step PCR products using i5 forward and i7 reverse primers. The amplified products were then purified as described for the first PCR round. The DNA was quantified using the QuantiFluor^® ONE dsDNA System (Promega), and the sample quality was confirmed using a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). The 16S rRNA libraries and amplified genes were sequenced using a MiSeq v3 Reagent Kit (Illumina, Inc.).

Affymetrix Cytogenetics Whole-Genome 2.7 M Microarrays and CytoScan™ HD arrays carry 2,700,000 unique, non-polymorphic probes covering the whole genome for detection of 35 kb or higher copy number variants as well as runs of homozygosity (ROH). Analysis was performed according to manufacturer protocols (Affymetrix Inc., Santa Clara, CA). Briefly, 3 μl of genomic DNA (33 ng/μl) are denatured and neutralized, and then amplified by PCR. The PCR products are then purified using magnetic beads (Beckman Coulter, Beverly, MA). The purified PCR products are fragmented and end-labeled with biotin. The fragmented, labeled PCR products are then hybridized overnight to the arrays. The arrays are washed and stained using the GeneChip^® Fluidics Station 450, and DAT images are acquired using the GeneChip^® Scanner 3000 (Affymetrix Inc.). Microarray data are available in the ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-4963. Data analysis is performed using Affymetrix Chromosome Analysis Suite (CHAS). Pathway Studio software version 9.0 (Ariadne Genomics, Rockville, Md., USA) was used to identify pathways and biological processes common to genes within ROH regions and place them in networks related to neuronal disease or autism.