Bigdye cycle sequencing kit

Directed mutagenesis was performed using the QuickChange Site-Directed Mutagenesis Kit (Stratagene). Oligonucleotides containing the mutation (Key Resources Table) were designed following the manufacturer’s recommendations. 2.5 U of PfuTurbo DNA polymerase was added to each sample and they were amplified by performing the following cycles: 1 denaturation cycle at 95 ° C for 1 min, 18 amplification cycles of 30 s at 95 ° C, 50 s at 60 ° C, 1 min / kilobase of plasmid at 68 °C and the last extension cycle 7 min at 68°C. Once amplified, 10 U of Dnp I enzyme were added and incubated at 37 ° C for 1 hr to digest the supercoiled double-stranded DNA and eliminate the parental molecules that are not mutated.
The correct introduction of the mutation was evaluated by plasmid sequencing using the BigDye Cycle Sequencing Kit (Applied Biosystems).

PCR reactions to obtain fragments used in cloning procedures or in screening of
transformed clones were carried out with Pwo (Roche) or similar high fidelity
DNA polymerases, respectively, as previously reported58 (link).
Sequences of primers used in amplification reactions are listed in Table S1. All amplified PCR products and
plasmids were sequenced by using the Big Dye cycle sequencing Kit (Applied
Biosystems) in an automated DNA sequencer (Applied Biosystems, 3130xl genetic
analyser).

Detection of noroviruses and sapoviruses by real-time RT-PCR has been described in detail elsewhere [11 (link)]. Positive specimens were genotyped by conventional RT-PCR [14 (link), 15 (link)] and PCR products of appropriate size (norovirus GI: 330 base pairs, norovirus GII: 344 base pairs, sapovirus: 434 base pairs) were gel-purified using the QIAquick Gel Extraction Kit (Qiagen). Sanger sequencing was performed using the Big Dye Cycle Sequencing Kit (Applied Biosystems) and sequences (available upon request) were genotyped by comparison to norovirus and sapovirus reference strains in the database of the National Calicivirus laboratory at CDC. Phylogenetic analyses were performed using MEGA (version 6.0) and statistical analyses were performed in Stata (version 13).
The Institutional Review Boards of the Navajo Nation, Phoenix Area Indian Health Service, Centers for Disease Control and Prevention and Johns Hopkins Bloomberg School of Public Health approved this research, as did the Navajo and White Mountain Apache tribes. Parents provided written consented for participation of their infants in the original rotavirus vaccine trial.

DNA from skin samples was extracted using a standard proteinase K/phenol/chloroform protocol [48 ]. Buffers used for extraction, precipitation and elution of DNA from blood and skin tissue are listed elsewhere [24 (link)]. DNA from the Callitrichid Research Center samples was extracted at Arizona State University (ASU). DNA from Brasília individuals was extracted at Northern State Fluminense University, Rio de Janeiro State, Brazil, and then exported to ASU (CITES permit #11BR007015/DF). DNA from all other individuals was extracted at the Federal University of Viçosa (UFV), Viçosa, Minas Gerais, Brazil.
Mitogenomes were obtained for a subset of the samples (Table S1) following the long-range PCR (LR-PCR) methodology of [24 (link)], and sequenced on an ABI 3730 sequencer with the BigDye Cycle Sequencing Kit (Applied Biosystems) by the ASU School of Life Science DNA Core Laboratory. The remainder of mitogenomes was obtained from whole genome sequencing (WGS). Individual WGS sequencing libraries were prepared at UFV and ASU with Illumina Nextera DNA Flex Library Prep Kits (catalog #20018704) following manufacturer’s instructions. Individual libraries were barcoded with Illumina Nextera DNA CD Indexes (catalog # 20018707), and pooled in equimolar amounts and sequenced on an Illumina NextSeq using v2 chemistry for 2 × 150 cycles at the ASU Genomic Core Facilities.

The PCR product was purified using the ExoSAP clean-up kit (Thermofisher Scientific, USA). The PCR amplicons were then sequenced using the BigDye Cycle Sequencing Kit (Applied Biosystems, USA). The sequencing was performed with the same forward primers as were used for the RT-PCR [43 (link)]. After the sequencing reaction, an ethanol precipitation was performed and the final product was loaded in ABI PRISM 3130 automated sequencer (Applied Biosystems, USA) [44 (link)].