Truseq nano dna

Libraries were prepared consistently across three sequencing centers (Annoroad (ARD), NovoGene (NVG) and WuXi NextCODE (WUX)) using 200 ng of DNA with the Illumina TruSeq DNA nano following manufacturer’s instructions. DNA fragmentation was achieved using a Covaris (LE220) instrument with a target size of 350bp. All libraries were assessed for quantity and quality using the Qubit 3.0 fluorometer with the Quant-iT dsDNA HS Assay kit (ThermoFisher Scientific, Q32854) and the Agilent 2100 Bioanalyzer or TapeStation instrument. All materials were prepared with three replicates in a single batch at each sequencing center. They were then sequenced on the Illumina X10 platform with paired end 150 bp read length leveraging synthesis (SBS) chemistry per the manufacturer’s instructions.

Genomic DNA (100 ng) was fragmented at 350 bp by ultrasonication. Libraries were constructed following the TruSeq DNA Nano protocol (Illumina). In brief, sheared gDNA was end-prepped, dA-tailed and enriched for fragments of ~350 bp through size selection with AMPure XP beads (Beckman Coulter). Indexing PCR for a total of eight cycles was performed for the size-selected fragments, and the products were purified with AMPure XP beads. The quality of the final libraries was checked on the LabChip GX Nucleic Acid Analyzer (PerkinElmer). High-coverage sequencing was performed on the Illumina NovaSeq 6000 system with the S4 flow cell and PE150 configuration. Further details can be found in the Supplementary Materials.

Genomic DNA was extracted from whole blood or saliva using a Qiagen (Valencia, CA) QIAamp DNA Maxi kit or a prepIT L2P kit (DNA Genotek Inc., Ontario, Canada), respectively. Probands underwent chromosomal microarray testing on Illumina (SanDiego, CA) platforms, with the reportable effective resolution of arrays being 200 Kb. Results were analyzed with Karyostudio software version 1.3 or 1.4 (Illumina), using genome reference sequence NCBI36/hg18 (v1.3, pre-2013) or GRCh37/hg19 (v1.4, 2013 onwards).
Genome sequencing was conducted on 204 individuals from 70 families comprising 71 probands (two probands were monozygotic twins hence for the genetic analysis and results we report on 70 probands), 127 parents and 6 other relatives. Illumina TruSeq DNA Nano or NovaSeq PE150 PCR free library preparation was completed prior to sequencing on the Illumina NovaSeq 6000 to average 30-fold depth with ~100 Gb data generated per sample at the Australian Genome Research Facility or Novogene (HK) Company Limited. Sanger sequencing or droplet digital PCR (ddPCR) were used to segregate variants in additional family members who had not undergone microarray or genome sequencing.

DNA was extracted from cells using a DNeasy Blood & Tissue kit (Qiagen, 69504) following the manufacturer’s protocol. Library preparation was performed using TruSeq DNA PCR-Free (Illumina, 2001596) or TruSeq DNA Nano (Illumina, 2001596) kits. Libraries were sequenced on a NovaSeq 6000 system (Illumina) with a PE150 configuration.

Total DNA was used to construct paired-end libraries (150 bp) according to the TruSeq™ DNA Nano protocol and sequenced on the Illumina Novaseq platform (Macrogen, Republic of Korea). The quality of the raw reads was assessed using FastQC (https://github.com/s-andrews/FastQC) and then trimmed using Trim Galore (https://github.com/FelixKrueger/TrimGalore) with default settings.

Library preparation and sequencing were outsourced (https://www.fasteris.com/dna/). Briefly, DNA was converted into a double-stranded library using a custom TruSeq DNA Nano Illumina protocol omitting the fragmentation step and using a modified bead ratio to keep small fragments. Sequencing of both HERB_1937 and the negative control sample was performed in a paired-end 2×150 cycles configuration on a single lane of the NextSeq flow cell.