3500xl genetic analyser

All C. difficile isolates, received or cultured by the national reference laboratory, were characterised with PCR ribotyping. PCR ribotyping was performed using capillary gel electrophoresis (3500xL Genetic Analyser of Applied Biosystems, USA).⁷ To determine the genetic relatedness of strains with the same ribotype, MLVA was performed as described previously.⁵ (link) A minimum spanning tree was constructed to determine the genetic distance between isolates, based on the number of differing loci and the summed tandem repeat difference (STRD) using BioNumerics version 7.6.3 (Applied Maths). Isolates belonged to a clonal complex or genetic cluster when there was an STRD ≤2.⁸ (link) The five most common ribotypes (RTs) among all ribotyped isolates in 2020 were assessed and the percentage of these ribotypes among all ribotypes were compared between 2020 and 2015 through 2019. MLVA was performed on stored isolates with ribotypes that were more or less common in 2020 compared to previous years. These isolates were from the whole pandemic period and the same calendar period in 2015 through 2019 or, for RT014, for the pandemic period and 2019 only due to the large number of isolates.

Direct sequencing of exons 18 to 22 from EGFR (NM_207655) and exon 2 from KRAS (NM_021284) was performed to identify possible genetic alteration related to lung carcinoma in the LLC cells.
Genomic DNA from LLC cells, tumors and adjacent tissue were purified using DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.
Samples were amplified using GoTaq® Flexi DNA Polymerase (Promega, Wisconsin, USA). For PCR and sequencing primers refer to Table 2. PCR products were cleaned up using ExoSAP-IT (Affimetrix, California, USA) and sequenced in both directions using BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, California, USA). Sequencing reactions were run on a 3500xL Genetic Analyser (Applied Biosystems, California, USA) and were analyzed with Geneious R9 [34 (link)].

Sequencing was performed using different sets of primers for the S, M and L segments as defined above using Big Dye V3.1 kit (Applied biosystems) and injection on a 3500XL genetic analyser (Foster city, California, USA). The sequences obtained were cleaned and edited using Bioedit software (www.mbio.ncsu.edu/BioEdit/BioEdit.html), USA for both the reads from the forward and reverse primers. Sequences obtained were compared to those in the gene bank using the Basic Local Alignment Search tool (BLAST) [17] (link) in NCBI GenBank (http://www.ncbi.nlm.nih.gov/blast/Blast) to identify similar sequences. The clean sequences of each segment of each phenotype were aligned against the corresponding segment sequences of the wild type virus isolate using Bioedit. Nucleotide and amino acid similarity and diversity between the virus phenotypes were computed in MEGA v5.20 [18] (link) using the p-distance method.

The QIAamp Viral RNA Mini kit (catalogue no.52906, Qiagen, Hilden, Germany) was used to extract RNA from the throat swab samples. Reverse-transcription PCR (RT-PCR) was performed using primers amplifying the full-length G gene of HMPV [4 (link)] and Superscript III Platinum™ One-Step qRT-PCR Kit (catalogue. No. 1821198, Invitrogen, Thermo Fisher Scientific, Carlsbad, CA 92,008, USA). The reverse transcription was done at 50 °C for 30 min followed by initial denaturation at 95 °C for 15 min, then 40 cycles of 94 °C for 1 min, 52 °C for 1 min, and 72 °C for 1 min. A final extension was done at 72 °C for 10 min. The PCR products were subjected to agarose gel electrophoresis and observed under UV-transilluminator. The positive PCR products (n = 20) were purified using GenElute™ Gel Extraction Kit (Lot No. SLBZ2166, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). The purified products (n = 20) were sequenced using BigDye Terminator (version 3.1) cycle sequencing kit (Applied Biosystems, Foster, CA) according to the manufacturer's protocol. Purified sequences were analysed using a 3500xL Genetic analyser (Applied Biosystems, Foster City, California 94,404, USA.). All purified products were sequenced using both HMPV forward and HMPV reverse primers.

After amplification, 1 μl of the product or 1 μl of ladder were combined with 9 μl Hi-Di formamide and 0.3 μl internal size standard. The 19 STR genotypes were conducted by capillary electrophoresis on Applied Biosystems™ 3500xL genetic analyser with default instrument settings. Subsequently, the raw data and the allelic ladder were analysed by GeneMapper ID-X software. All sizes were calculated by using internal size standards on the 70, 80, 100, 125, 150, 175, 200, 233, 266, 300, 333, 366, 400, 445, 490, and 500 bp.

To examine diversity between locations, psyllids were DNA barcoded using one or two gene regions. The intergenic spacer 2 (ITS2) and cytochrome c oxidase subunit 1 (CO1) were amplified and sequenced for identification to species. For amplification of ITS2 primers CAs5p8sFcm-F and CA28sB1d-R^{65 (link)} were used and for amplification of CO1 gene regions arthropod barcoding Primers LCO1490 and HCO2198⁶⁶ were used. PCR amplified gene regions were sequenced using the Big-Dye Terminator Cycle Sequencing Kit (Applied Biosystems), forward and reverse complementary DNA strands were sequenced for each sample and analysed using a 3500xL Genetic Analyser (Applied Biosystems). Closest matches were found using BLAST on the GenBank database, BOLD database and our own in-house psyllid barcoding database. Specimens with ≥ 99% similarity were deemed the same species. Specimens that did not match sequences in any of the three databases were identified morphologically.

Fresh-frozen and paraffin-embedded tissue samples were used for RNA extraction using MagMAX Viral RNA Isolation Kit (Thremo Fisher Scientific). Reverse transcriptase polymerase chain reaction (RT-PCR) for morbillivirus was conducted with extracted RNA as described previously [10 ,30 ] using the SuperScript III One-Step-RT-PCR System (Invitrogen). The positive PCR amplicons were subsequently sequenced by using the ABI BigDye Direct Sequencing method in Applied Biosystems 3500 xL Genetic Analyser (Applied Biosystems, USA).