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Page 101 Chu et al. J Transl Genet Genom 2023;7:196-212 https://dx.doi.org/10.20517/jtgg.2023.22

automation sequencing workflow; and (iv) quality assurance/quality control measures, privacy protection,
and electronic records. By documenting the experiences and challenges in designing the laboratory and
establishing the GS workflow in a tight timeline, lessons learnt might assist international counterparts in
steering their own course in the genomic medicine era.

METHODS
Participant recruitment
The HKGP starts its operational workflow with the participants’ journey, from engaging referring clinicians
in recruitment to ending the diagnostic odyssey with personalised treatment by offering the first end-to-end
GS service in Hong Kong [Figure 1]. Eligible patients and families are recruited by a HKGP team set up in
each hospital (known as the Partnering Centre; PC). The University of Hong Kong/Queen Mary Hospital
(HKU/QMH; HKWC), the Chinese University of Hong Kong/Prince of Wales Hospital (CUHK/PWH;
NTEC), and the Hong Kong Children's Hospital (HKCH; KCC) are PCs in the Pilot Phase. In the main
phase, recruitment has been extended to other cluster institutions of Hong Kong West Cluster (HKWC,
namely The Duchess of Kent Children's Hospital at Sandy Bay (DKCH) and Grantham Hospital (GH) and
New Territories East Cluster (NTEC, namely North District Hospital (NDH) and Alice Ho Miu Ling
Nethersole Hospital (AHNH). Eligible participants are referred to PCs by clinicians after screening and
[7]
informed consent is conducted through face-to-face interviews .
Sample collection and transfer
For each participant, 6 mL of blood is obtained and stored in two 3-mL EDTA-containing anticoagulation
tubes. RapidDri Pouch kit (Isohelix) is used to collect cells inside the cheek for buccal swabs, and saliva
samples are collected using the GeneFix Saliva DNA Collection and Stabilization Kit (Isohelix). Specimen
collection is performed at the PCs. The specimens are packed in zip bags and stored at 4 °C until being
transferred to the HKGI Laboratory, or for a maximum of 72 h. The specimens are packed with cooling
packs and temperature-logging devices in the isothermal transfer boxes with combination locks.

Genomic DNA extraction and QC
Following sample registration, blood samples were aliquoted either manually or using the liquid handling
system Freedom EVO100 (Tecan). Genomic DNA (gDNA) was extracted from 400 µL of whole blood using
the QIAsymphony SP system and QIAsymphony SP DNA Midi Kit (Qiagen). For saliva and buccal swab
samples, gDNA was extracted using the EZ2 Connect system and EZ1&2 DNA Tissue Kit (Qiagen). gDNA
concentration was determined using the Qubit dsDNA BR assay kit and was measured with the Qubit 4
Fluorometer (Thermo Fisher Scientific). gDNA purity was determined using a NanoDrop One
Spectrophotometer (Thermo Fisher Scientific). gDNA integrity was assessed for degradation using a 1%
E-Gel precast gel electrophoresis system (Thermo Fisher Scientific). Approximately 100 ng of each gDNA
sample was loaded into the precast agarose gel.

Illumina GS sequencing and QC
PCR-free GS libraries were constructed using the KAPA HyperPlus kit for PCR-free workflow and KAPA
Unique Dual-Indexed adapter kit (Roche) following the instructions provided by the manufacturer. 1 µg of
gDNA is fragmented enzymatically at 37 °C for 15 min, end-repaired, 3′dA-tailed, ligated to dual-index
adapters, and size-selected. Reaction cleanup and double-sided size selection steps were performed using
KAPA HyperPure Beads (Roche). For the double-sided size selection, 50 µL of beads were added to the
adapter-ligated library to remove large-sized DNA fragments in the first cut. To remove small-sized DNA
fragments, 8 L of beads were added to the supernatant from the first size cut, resulting in the final library
size range of 400-700 bp.