Bigdye terminator version 1.1 cycle sequencing kit

Validation of key variants identified was performed using Sanger sequencing. Regions of interest were PCR amplified and sequenced using a BigDye Terminator version 1.1 cycle sequencing kit (Applied Biosystems, Foster City, CA) on an ABI 3130 sequencer (Applied Biosystems), and visualized using Sequencher version 5.2.4 (Gene Codes Corporation, Ann Arbor, MI). Control data for population genomic variation was obtained using the Genome Aggregation Database (gnomAD) (gnomAD, Cambridge, MA, https://gnomad.broadinstitute.org) [24 ].

Genomic DNA was extracted from heat inactivated individual bacterial colonies using the High Pure PCR template preparation kit (Roche Applied Sciences, Mannheim, Germany) according to the manufacturer's instructions. AMOS-PCR for B. abortus, B. melitensis, B. ovis, and B. suis was done as previously described [7 (link), 10 (link)]. Partial 16S rRNA genes of the bacterial isolates were amplified by PCR with the 16SUNI-L (5′-AGA GTT TGA TCA TGG CTC AG-3′) and 16SUNI-R (5′-GTG TGA CGG GCG GTG TGT AC-3′) primer pair (Jena Bioscience GmbH, Jena, Germany) to generate amplicons of approx. 1,400-bp [11 (link)]. PCR products were analyzed by agarose gel electrophoresis, bands were cut out, and DNA was purified using a Gel Extraction Kit (Qiagen, Hilden, Germany) according to the manufacturer's recommendations. Cycle sequencing of the partial 16S rRNA genes was done in both directions by using forward and reverse amplification primers with a BigDye Terminator Version 1.1 Cycle Sequencing Kit (Applied Biosystems, Darmstadt, Germany) according to the recommendations of the manufacturer. Sequencing products were analyzed with an ABI Prism 3130 Genetic Analyzer (Applied Biosystems). Identification of isolates was done by a BLAST search (https://www.ncbi.nlm.nih.gov/blast/) using 16S rRNA gene sequences.

Genomic bisulfite-treated DNAs from HCC cell lines were sequenced. PCR was conducted in the COL1A1 promoter region for the sequencing. The PCR primer pairs were S (5′-GGGTAGGGTTTTTTTTTGTTTTT-3′) and AS (5′-CTAAACCCTAAACATATAAACTC-3′), which amplify a 179-bp product. PCR amplification consisted of 35 cycles of 94°C for 15 s, 51°C for 12 s, and 72°C for 12 s after the initial denaturation step (94°C for 5 min). PCR products were purified directly using the QIAquick PCR Purification Kit (Qiagen). Finally, purified templates were prepared for direct sequencing using the BigDye Terminator version 1.1 Cycle Sequencing Kit (Applied Biosystems) and the BigDye Xterminator (Applied Biosystems). Sequence analysis was carried out using an Applied Biosystems ABI310, and sequence electropherograms were generated using ABI Sequence Analysis software version 5.1.1.

Parasite DNA was isolated from the dried blood spots on filter paper (Advantec, Toyo Roshi Kaisha Ltd, Japan) using QIAamp DNA blood mini kit (Qiagen, the Netherlands) as described by Sakihama et al.[27 (link)]. Plasmodium falciparum was identified by nested PCR technique using primers of Snounou et al. and Kimura et al.[28 (link),29 (link)]. Pfdhfr and pfdhps genotypes were determined by sequence analysis as previously described by Isozumi et al.[30 (link)]. The amplified PCR products were directly used as templates for sequencing with a BigDye Terminator (version 1.1) cycle sequencing kit and a model 3730 genetic analyzer (Applied Biosystems, USA). Alleles at residues 16, 51, 59, 108, and 164 of the pfdhfr, and at residues 436, 437, 540, 581, and 613 of the pfdhps were read carefully, and two independent PCR products were subjected to sequence analysis in the case of new or rare mutations.

Viral RNA of the 272 novel sequences was extracted from original biological samples (n = 261) and growth medium after passage in cell culture (n = 11). Reverse transcription and PCR assays targeting a 288 bp region of 5′UTR of BVDV were performed using previously described primers by Letellier et al. [31 (link)], with the exception of strains collected in Sicily, which were tested using primers by Vilcek et al. [32 (link)]. The samples collected in the Center and the South macroareas were also tested by primers for atypical Pestivirus [14 ]. For the BVDV-1 subtypes identified for the first time in Italy, a 428 bp region encoding autoprotease N^pro was amplified using nested PCR, as previously described [20 (link), 29 (link)].
For each sample, the amplicons of the expected sizes were purified and sequenced using forward and reverse primers by cycle sequencing using a Big Dye Terminator version 1.1 Cycle Sequencing kit (Applied Biosystems, CA, USA) and an ABI PRISM 3130 sequencing device or sent for outsource sequencing (Primm).