Ion pgm instrument

Genomic DNA was purified from four representative ST-1562 strains that were positive for tetO with the 23S rRNA mutation (A2085G) (CCP005, CCP013, CCP014, and CCP022) by Genomic-tip 100/G in combination with the Genomic DNA buffer set (Qiagen), and was then used to construct libraries with the Ion Xpress Plus Fragment Library kit and Hi-Q View Chef 400 kit using the Ion Chef System (Thermo Fisher Scientific). The libraries on Ion 316 Chip v2 BC were then subjected to Ion semiconductor sequencing with the Ion Hi-Q View Sequencing kit in an Ion PGM instrument (Thermo Fisher Scientific). Sequence data (4,099,697, 2,339,519, 2,783,415, and 2,565,055 reads for CCP005, CCP013, CCP014, and CCP022, respectively) were assembled using CLC Genomic Workbench ver. 9.0 (Qiagen). Totals of 185, 70, 123, and 124 contigs were obtained (>500 bases) for the strains CCP005, CCP013, CCP014, and CCP022 at 516-, 335-, 332-, and 282-fold coverages, respectively. Whole genome shotgun sequence data were deposited at DDBJ/EMBL/GenBank under the accession numbers BHEH01000000 (CCP005), BHEI01000000 (CCP013), BHEJ01000000 (CCP014), and BHEK01000000 (CCP022).

We designed LR-PCR primers to amplify 18 long DNA fragments, including the exonic regions of PKD1 and PKD2. These fragments were amplified from participants’ purified genomic DNA using the primers shown in S1 Table. Multiple LR-PCR products were amplified in each of the following combinations: A (LR-PCR 2 and 12), B (4 and 17), C (6 and 15), D (1, 5, and 9), E (3, 8, and 18), F (7, 13, and 16), and G (10, 11, and 14). The LR-PCR reactions were performed simultaneously on the same PCR plate using the following touchdown PCR regimen [19 (link)]: (i) 94°C for 2 min; (ii) one cycle at 98°C for 10 s and 74°C for 5 min; (iii) one cycle at 98°C for 10 s and 72°C for 5 min; (iv) one cycle at 98°C for 10 s and 70°C for 5 min; (v) 30 cycles at 98°C for 10 s and 68°C for 5 min; and (vi) 68°C for 7 min. The LR-PCR products were purified using the Agencourt AMPure XP kit (Beckman Coulter, Inc, Brea, CA, USA) and quantified as previously described [20 (link)].
With the Ion PGM instrument (Thermo Fisher Scientific Inc, Waltham, MA, USA), only weak coverage could be achieved for the PKD1 exon 1 region because it contains a GC-rich sequence. To circumvent this problem, corrective PCR was performed in which PKD1 exon 1 was re-amplified from the combination D LR-PCR product noted above, using the primers shown in S2 Table. The PCR products were purified according to the procedure described above.