Abi genetic analyzer

The RBIP primers were designed by Primer3⁴⁰ . One primer was designed from LTR sequence and another was designed from flanking genome sequence. The design principles were as follows: (1) the primer length was 18–25 bp; (2) the amplified products were 100–1000 bp; (3) the GC content of the primers was 35–55%; (4) the annealing temperature was 50–60 °C; and (5) the annealing temperature difference between upstream and downstream primers was less than 5 °C. The designed primers were synthesized by Beijing Tsingke Biotechnology Co., Ltd.
PCR amplification was carried out in 15 μL reaction solution consisting of 1 μL DNA template, 7.5 μL Tsingke Master Mix (Tsingke, Beijing, China), 1 μL (10 μmol L–1) of each RBIP primer (Tsingke, Beijing, China) and 4.5 μL deionized distilled water. PCR amplification was performed with the following procedure: 94 °C for 5 min followed by 35 cycles at 94 °C for 30 s, 58–60 °C (depending on the RBIP primers) for 30 s, 72 °C for 30 s, a final extension at 72 °C for 5 min, and storage at 4 °C. Amplicons were analyzed by electrophoresis on a 2% (w/v) agarose gel. Amplicons were pooled together with an internal size standard (ABI GeneScanTM 500 LIZ, Applied Biosystems, Foster City, USA) and loaded on an ABI Genetic Analyzer (3730XL, Applied Biosystems, Foster City, USA). Accurate allele points were analyzed by Gene mapper 4.1 software⁴¹ .

Primer pairs used for RT-PCR were designed using the bovine coding sequences and software available from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov); preferably, each primer of a pair was located on a separate gene exon (Table 1). All PCR products were sequenced (ABI Genetic analyzer, Applied Biosystems, Foster City, CA, USA) to verify specificity.

DNA extracts from N. gonorrhoeae isolates were obtained with the QIAmp DNA extraction kit (Qiagen, USA), according to manufacturer’s instructions. NG-MAST and sequencing of penA were performed as previously described [10 (link), 21 (link), 22 (link)]. Purified PCR products were sequenced with an ABI genetic analyzer (Applied Biosystems, Perkin Elmer USA).

Individual M. cinereus specimens were collected by a commercial trawl fishing method in Zhoushan City, Zhejiang Province, China (30° 40′ 30″ N, 121° 20′ 28″ E) and immediately preserved with 95% ethanol. Total genomic DNA was extracted using the SQ tissue DNA kit (OMEGA) according to the manufacturer's protocol. After extraction, the DNA was stored in − 4 ℃ refrigerator. The polymerase chain reaction (PCR) primers used in this experiment designed 10 pairs of primers for the amplification of the complete mitochondrial genome of M. cinereus based on the complete mitochondria published by the predecessors^{13 (link),69 (link),70} (Table S1). The PCR was carried out in a 25 μl reaction volume containing 2.0 mM MgCl₂, 0.4 mM of each dNTP, 0.5 μM of each primer, 1.0 U of Taq polymerase (Takara, China), 2.5 μl of 10 × Taq buffer, and approximately 50 ng of DNA template. Using the following cycling conditions: (1) initial activation step for 5 min at 95 °C; (2) 35 cycles of denaturation at 95 °C for 30 s, annealing at 52 °C (as the case may be) for 30 s and extension at 72 °C for 30 s; and (3) a final extension of 5 min at 72 °C. The sequences were determined using an ABI genetic analyzer (Applied Biosystems, China).

Sanger sequencing was performed to confirm all detected variants. The primers Prism 3130 were designed using the online software Primer3. The amplification reaction mixture (50 μl) was subjected to denaturation at 95 °C for 2 min followed by 30 cycles at 94 °C for 1 min, annealing temperature 60 °C for 1 min, 72 °C for 1 min and by a final extension at 72 °C for 15 min. Bi-directional sequencing was performed using BigDye Terminator v3.1 Cycle Sequencing Kit, version 3.1 (Applied Biosystems, Foster, CA, USA) and analyzed on an ABI genetic analyzer (Applied Biosystems, Foster City, CA, USA).

The microbiota fingerprints of all gut samples in this study were obtained using DGGE as described in da Mota et al. [26 (link)] with the D-code system (Bio-Rad Laboratories, Munich, Germany). Amplicons of bacterial 16S rDNA were loaded onto a 6% polyacrylamide gel that was prepared with a denaturing gradient in the range of 45% - 65%. The denaturing agent (100%) was a solution of urea (7 M) and formamide (40%). Electrophoresis was run in TAE (Tris acetic EDTA) buffer at 60°C for 16 h at 75 V. After electrophoresis, gels were stained with SYBR Green I (Sigma Aldrich, MO, USA) and subsequently digitized using a Thyphoon Trio scanner (GE Healthcare Life Science, USA) with 400 nm excitation and 520 BP emission filters. Dominant bands (thicker bands) were excised from DGGE gels and transferred to 1.5 ml tubes. The gel slices were crushed with the top of a sterile tip and eluted in 50 μl of water for sequencing. The eluent was used as PCR template DNA using primers U968 and L1401. DNA sequencing was performed with an ABI Genetic Analyzer (Applied Biosystems, CA, USA). The partial 16S rDNA sequences from DGGE bands were assigned to uncover the taxa positions of bacteria until the genus level using the SeqMatch program [37 (link)] of the Ribosomal Database Project (RDP) to preliminarily identify bacterial microbiota members.

The sample of G. dengba was collected in Chayu City, Tibet, China (28°66′ N, 97°46′ E). Samples of G. tibetana and G. yajiangensis were collected in Motuo City, Tibet, China (29°32′ N, 95°33′ E). Total genomic DNA was extracted from muscle tissue using the Qiagen QIAamp tissue kit according to the manufacturer’s protocol. The DNA sediment was solubilized in double-distilled water, stored at 4°C, and then quantified in concentration. The G. tibetana mitogenome was amplified with ten pairs of universal primer by general PCR. The general PCR cycle requirement for DNA amplification is 5 min at 94°C, [30 s at 94°C, 30 s at 55–56°C, 1 min at 72°C] X 35 cycles, and 10 min at 72°C. Sequences were sequenced using an ABI Genetic Analyzer (Applied Biosystems, China). Complete mitogenome sequencing of G. dengba and G. yajiangensis was performed on an Illumina HiSeq X Ten platform.