Abi bigdye terminator v3.1 cycle sequencing kit

Genomic DNA was extracted from paraffin-embedded tumor tissues using QIAamp® DNA Mini Kit (Qiagen, Germany), according to the manufacturer's instructions. Quality and concentration of the DNA samples were examined by NanoDrop (Thermo). The primers used to amplify BRAF exon 15 were as follows: forward 5′-TCATAATGCTTGCTCTGATAGGA-3′ and reverse 5′-GGCCAAAAATTTAATCAGTGGA-3′. Polymerase chain reaction (PCR) was carried as following: a final volume of 25 μl containing purified genomic DNA (100 ng/μl) 1 μl, 10 × ABI buffer 2.5 μl, MgCl₂ (25 mM) 1.5 μl, dNTP (2.5 mM) 2 μl, ABI AmpliTaq Gold DNA Taq polymerase 0.125 μl (5 U/μl), forward primer and reverse primer (10 μM) 1 μl, after denaturation at 95°C for 10 minutes, 38 amplification cycles at 95°C for 30 s, 56°C for 30 s, 72°C for 45 s, and elongation at 72°C for 10 minutes. The PCR products sequencing were performed with ABI BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) according to the manufacturer's instructions. The sequencing primers were the same as the PCR primers. Sequencing reactions were electrophoresed on an ABI 3500XL genetic analyzer (Applied Biosystems). Sequence data were analyzed using an ABI 3500XL DNA Analyzer (Applied Biosystems).

The HBV S gene was detected by nested PCR as described previously [25 (link)]. HBV DNA was extracted from about 30 mg (obtained from approximately 20 sections, 5 μm thick, not attached to glass slides) of paraffin-embedded synovium with the RecoverAll™ total nucleic acid isolation kit (Life Technologies). Liver tissue from patients with HBV-related hepatocellular carcinoma was included as a positive control. Amplification was carried out in a 50-μl reaction volume containing 3 μl of forward and reverse primers (10 μM), 40 ng of DNA template, and 25 μl of 2 × KAPA HiFi HotStart ReadyMix (Kapa Biosystems, Wilmington, MA, USA). The following thermocycles were used: 95 °C for 3 minutes, followed by 35 cycles of 98 °C for 20 seconds, 65 °C for 15 seconds, and 72 °C for 1 minute, with a final extension at 72 °C for 1 minute. The PCR products were then resolved by gel electrophoresis (Life Technologies). DNA bands were visualized by ultraviolet fluorescence. PCR products were sequenced in both directions on an ABI 3730 XL Automated DNA Sequencer with the ABI BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA). The sequences were aligned using the Basic Local Alignment Search Tool (National Center for Biotechnology Information website https://blast.ncbi.nlm.nih.gov/Blast.cgi) to confirm the identity of the HBV S gene.

Genomic DNA was extracted from the patient’s blood samples using DNA Quick II (Genomic DNA Isolation kit; DS Pharma Biomedical Co., Ltd.). PCR amplification was performed using the primers whose sequences are listed in Supplementary Table 1. Primers were designed from the NCBI Probe database. For PCR, 0.4 µL template DNA, 0.4 µL of 10 µM forward and reverse primers, 1.6 µL dNTP, 2.0 µL of 10 × Ex Taq buffer, and 0.1 µL Ex Taq HS were mixed with 15.1 µL sterile water. The thermal cycling conditions were as follows: preamplification denaturation (1 cycle), 94 °C for 30 s; amplification (total of 30 cycles), 98 °C for 10 s, Table 1 listed temperature for 30 s, 72 °C for 30 s; and final elongation (1 cycle), 72 °C for 2 min. PCR products were purified with illustra ExoStar (GE Healthcare Life Sciences). The purified PCR products were used for sequencing with an ABI BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) using M13 forward or reverse primers. After ethanol precipitation, automated sequencing was performed on an ABI 3100 Genetic analyzer using both the forward and reverse primers. Variants found in dbSNP, the 1000 Genomes Project database, or the Exome Variant Server database of the National Heart, Lung, and Blood Institute Exome Sequencing Project were excluded.

PCR products were sequenced using the forward and reverse primers of the secondary PCR. An intermediary sequencing primer gp60-R3 [5′-GAG ATA TAT CTT GTT GCG-3′] was also used in the sequencing of gp60 PCR products. DNA sequencing was done using the ABI BigDye Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and an ABI3130 Genetic Analyzer (Applied Biosystems). Sequence accuracy was confirmed by sequencing of two PCR products from each positive specimen. Nucleotide sequences obtained were aligned with reference sequences using the ClustalX 1.81 package (http://www.clustal.org/) to identify Cryptosporidium species and C. parvum subtypes. Subtypes of C. parvum and C. hominis were named based on the established nomenclature system [29] (link). Unique sequences generated in this study were deposited in GenBank under accession numbers AB830575 to AB830590.