La pcr kit ver 2

Long‐range PCR was performed using four primer pairs amplifying both SMN genes (NG_008691.1, NG_008728.1) in four overlapping PCR products (PCR1‐PCR4, Table 1). The reactions were performed with TaKaRa LA PCR Kit Ver. 2.1 according to the manufacturer`s protocol. PCR products were pair‐end sequenced on Illumina HiSeq 2500 (Illumina). FASTQ files were aligned to the human reference genome hg19 using NovoAlign (V2.08.03) and all alignment locations were reported. Picard Tools (1.129) were used to convert SAM to BAM, remove duplicates, and add read groups. Local realignment around indels, base recalibration, and genotyping was performed with the Genome Analysis Toolkit, GATK (3.5) (McKenna et al., 2010). Variants were annotated by SnpEff (4.3t) (Cingolani et al., 2012) and GEMINI (0.20.2‐dev) (Paila, Chapman, Kirchner, & Quinlan, 2013).

The full-length anellovirus was amplified by inverted nested PCR based on the sequence obtained from the MiSeq analysis. An overlapping PCR fragment including the remainder of the circular genome (containing the GC-rich region) was obtained and then sequenced. Amplification was performed using Takara LA Taq polymerase and GC buffer I (LA PCR Kit Ver.2.1, TaKaRa, Dalian, China) and was performed as follows: 94 °C for 3 min, followed by five cycles of 1 min at 94 °C, 1 min at 60 °C, and 3.5 min at 72 °C, and then 30 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 3.5 min, with an additional 1 s in the extension stage every cycle and a final extension step of 10 min at 72 °C. The second-round cycling conditions were the same as above. The PCR products were excised from 1% agarose gels containing ethidium bromide (0.5 g/ml) and were purified using an AxyPrep DNA Gel Extraction Kit (Axygene, Silicon Valley, USA) according to the manufacturer’s instructions.