Abi 3500 genetic analyser

To validate the disease-causing variants selected, Sanger sequencing was performed using specific PCR primers designed with Primer Premier 5. The sequences of the FBN2 primers used were FBN2-F: 5′-GCAAACTCACCAATACACTT-3′ and FBN2-R: 5′-CTCCATACGGTTGCATCTT-3′. The sequences of the COL1A2 primers used were COL1A2-F: 5′-GAACATGCTTCCGTGTGA-3′ and COL1A2-R: 5′-CATCAACTTCATAGTCCTTGG-3′. PCR products were sequenced using an ABI 3500 Genetic Analyser (Thermo Fisher Scientific) for COL1A2 c.3304G > C and FBN2 c.4108G > T.

The PowerPlex Y23 kit was used to generate 23 loci: DYS576, DYS389I, DYS448, DYS389II, DYS19, DYS391, DYS481, DYS549, DYS533, DYS438, DYS437, DYS570, DYS635, DYS390, DYS439, DYS392, DYS643, DYS393, DYS458, DYS385a/b, DYS456 and Y-GATA-H4. DNA amplification Reaction setup and thermal cycling were performed according to the procedures described in the PowerPlex Y23 kit User’s Manual. Thermal cycling was performed in Veriti^® 96-Well Thermal Cycler as recommended by the manufacturer (Thermo Fisher Scientific). PCRs were conducted using half of the recommended volume. Fragments were electrophoresed in eight capillaries (50-cm length) arrays on the ABI 3500 Genetic Analyser using the manufacturer’s recommended protocols (Thermo Fisher Scientific) filled with POP-4™ polymer. GeneMapper IDX software V1.4 was used for allele calling and interpretation.

Based on the results of the Respiratory Viruses 16-Well Assay V.17 and V.19, the remainder of 261 HRSV-positive stored NA samples were used to perform sequence analysis. Nested PCR was carried out with two sets of primers (0.4 µM of each primer in both reactions) and 1 µL of NA to amplify the second hypervariable region (HVR2) of the HRSV G gene using the primers and protocol described by Slovic et al. [12 (link)]. The nested PCR was performed by using the PrimeScript One Step RT-PCR kit (Takara Bio Inc., Shiga, Japan) in a Veriti™ 96-Well Fast Thermal Cycler (Thermo Fisher Scientific, Waltham, MA, USA) following the thermal cycling conditions as described previously [12 (link)]. The amplified products (501–573 bp for HRSV subgroup A and 560–564 bp for HRSV subgroup B strains) were detected by 1.5% agarose gel electrophoresis and sequenced by Sanger sequencing. Sequencing reactions were set up with Fast AP + Exonuclease I (Thermo Fischer Scientific, Waltham, MA, USA) enzymatically purified second-stage PCR products, using the primers as described by Slovic et al. [12 (link)] and BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Sequencing was performed on an ABI-3500 Genetic Analyser (Thermo Fisher Scientific, Waltham, MA, USA).