Abi3500 automated sequencer

Altogether, 73 randomly chosen isolates were characterized by the MLST scheme according to the B. cereus PubMLST database (https://pubmlst.org/bcereus/) and previous work (22 (link)). Briefly, 534- to 599-bp fragments of seven housekeeping loci (glpF, gmk, ilvD, pta, pur, pycA, and tpi) were sequenced using an ABI3500 automated sequencer (Applied Biosystems) according to a protocol previously described (22 (link)) (Table S2). The combination of allele numbers for all seven loci for a given isolate allowed assessment of the specific sequence type (ST). New allele sequences and STs were submitted to the B. cereus PubMLST database.
For phylogenetic analysis, altogether, 372 environmental isolates and 24 references were included. A phylogenetic tree based on the concatenated loci (2,829 nucleotides) was constructed for all isolates using the neighbor-joining (NJ) method together with MEGA7 software (53 (link)). Branch quality was evaluated using 1,000 bootstrap replicates. Detailed information about the strains used in this study is given in Table S1. The STs were assigned to clonal complexes using PHYLOViZ v2.0 (54 (link)) with the goeBURST algorithm and 1,000 bootstrap resampling according to Feil and coworkers (55 (link)). Clonal complexes (CCs) were defined as single locus variants (SLVs) of two or more independent isolates that shared identical alleles at six or seven loci.

Amplification products of the Babesia spp. 18S rDNA were purified using the QiaAmp PCR purification kit (Qiagen). Sequencing reactions of both strands were performed using Big Dye Terminator cycle sequencing kit (Applied Biosystems) with the F2 and R2 primers under the same conditions as above. Cycle sequencing reactions were purified using the ExTerminator Kit (A&A Biotechnology, Gdynia, Poland) and sequenced with the ABI3500 automated sequencer (Applied Biosystems, Foster City, USA). The obtained sequences were compared with sequences collected in the NCBI database (http://www.ncbi.nlm.nih.gov).

Two million PBMCs were treated with a lysis buffer containing Proteinase K and stored at − 20 °C for subsequent PCR amplification of a 372 bp C2-V5 HIV-1 env gene fragment comprising the V3 region (positions 7,001 to 7,339 relative to the HXB2; GenBank accession number K03455) with primers JA10/JA11 using PCR conditions previously described^{25 (link)}. The PCR products were purified with QIAquick purification columns (QIAGEN, Germany), and then sequenced using the DYEnamic ET Terminator Cycle sequencing kit v1.1 (Amersham Biosciences, England). Sequencing reactions were run on an ABI 3500 automated sequencer and analyzed with the Variant Reporter Software 2 (Applied Biosystems, USA). V3 loop sequences were identified within the HIV-1 C2-V5 env fragment. Amino acid V3 loop sequences are available as Supplementary Information.

We used the Sanger sequencing technique for the determination of β thalassemia mutations. PCR was used to amplify a fragment of 101 base pairs covering the coding region of the Beta Globin gene, which has approximately 619 base pairs, using the follow primers sequence: (P1) 5’-TCCTAAGCCAGTGCCAGAAG-3’ and the downstream primer (P5) 5’-TCATTCGTCTGTTTCCCATTC3’[13 ].
The purified PCR product was subjected to another PCR reaction using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems Foster City, CA) following the manufacturer's protocol. Subsequently, the products of this reaction were analyzed by an ABI3500 automated sequencer (Applied Biosystems).
The sequences were edited through Sequencher Software (GENECODES) and the results were classified as β⁺ or β⁰ thalassemia mutations previously described in the literature through an online tool HbVarDatabase, Inc. (http://globin.bx.psu.edu/hbvar/) [14 ,15 ].

The whole genomic DNA was extracted from freshly preserved (96% ethanol) scorpion specimens using Qiagen extraction kit (Qiagen) according to its manufacturer’s instructions. A fragment of COI gene was amplified via standard polymerase chain reaction (PCR) using invertebrate universal primers (LCO1490 and HCO2198) as determined by [29 (link)]. The amplified products of COI gene were checked, purified, and sequenced on an ABI 3500 automated sequencer (Applied Biosystems Inc., USA). The obtained sequences were deposited in GenBank (Table 1) at https://www.ncbi.nlm.nih.gov/genbank/ with accession numbers (OP970165, OP970166, OP970167, OP970168).

MSI assays were performed using paired normal and tumor samples as previously described^{47 (link)}. The five microsatellite markers including two mononucleotide repeats (Bat-25 and Bat-26) and three dinucleotide repeats (D2S123, D5S346, and D17S250) recommended by the National Cancer Institute were amplified in a single multiplex PCR reaction. The PCR products were analyzed by capillary electrophoresis using an ABI 3500 automated sequencer (Applied Biosystems, Foster City, CA). Two of the five microsatellite markers demonstrated instability, and the tumor was considered to be high MSI (MSI-H). MSI at a single locus was defined as low MSI (MSI-L), while the absence of instability at any of the markers was defined as microsatellite stable (MSS). For statistical purposes, MSI-L tumors were considered together with MSS tumors, because of the similarity between MSI-L and MSS tumors^{48 (link)}.