Bigdye v3.1 chemistry

MLST was performed via the PCR amplification of seven housekeeping genes (acbZ, bglA, cat, dapE, dat, ldh, and lhkA) per Institut Pasteur protocols (http://www.pasteur.fr/recherche/genopole/PF8/mlst/Lmono.html) until 2013. PCR products were purified using FastAP chemistry (Thermo Fisher Scientific, Waltham, MA, USA) and products sequenced on the ABI 3130x1 genetic analyzer using BigDye v3.1 chemistry (Applied Biosystems, Waltham, MA, USA). MLST profiles were analyzed in BioNumerics v7.5 (Applied Maths, Sint-Martens-Latem, Belgium) using the MLST plugin. Since 2012, in silico MLST has been inferred from WGS reads using SRST/SRST2 (v0.1.0 to v0.1.5) (7 (link)).

DNA was extracted from buffy coat and BAL using the QIAamp DNA Mini Kit (Qiagen). Single-genome amplification of a subgenomic HIV region (nef) was performed using primers designed to amplify major HIV subtypes [41 (link), 51 (link)]. Briefly, genomic DNA extracts were endpoint diluted such that <30% of the resulting nested polymerase chain reactions (PCR), performed using an Expand High Fidelity PCR system (Roche), would yield an amplicon. First round PCR primers were Nef8683F_pan (forward; TAGCAGTAGCTGRGKGRACAGATAG) and Nef9536R_pan (reverse; TACAGGCAAAAAGCAGCTGCTTATATGYAG). Second round PCR primers were Nef8746F_pan (forward; TCCACATACCTASAAGAATMAGACARG) and Nef9474R_pan (reverse; CAGGCCACRCCTCCCTGGAAASKCCC). Negative controls were included in every run. For the participants with archived plasma available, nucleic acids were extracted using the BioMerieux NucliSENS EasyMag system (BioMerieux, Marcy-l’Étoile, France). Next, cDNA was generated using NxtScript reverse transcriptase (Roche) using the reverse primer Nef9536R_pan, after which the cDNA was endpoint diluted and single-genome-amplified as described above. Amplicons were sequenced on a 3730xl automated DNA sequencer using BigDye (v3.1) chemistry (Applied Biosystems). Chromatograms were base called using Sequencher (v5.0/v5.4.6) (GeneCodes).

We used markers identified from the variable intergenic regions and ecoprimer to discriminate the four Cistanche species. Primers to discriminate between the four Cistanche species under study were designed on the variable intergenic regions using Snapgene 6.0 (Snapgene from Insightful Science, available at http://www.snapgene.com, last used on 2022), or were those selected with EcoPrimer software on the barcoding regions (Table S7 and S8). PCR amplifications were performed in a final volume of 20 μL with 10 μL 2 × Taq PCR Master Mix, 0. 5 μM of each primer, 5 μL template DNA, and 4 μL ddH₂O following the manufacturer’s instructions (Mei5 Biotechnology, Co., Ltd). All amplifications were carried out in a Pro-Flex PCR system (Applied Biosystems, Waltham, MA, USA) under the following conditions: denaturation at 95 ℃ for 3 min, followed by 36 cycles of 94 ℃ for 25 s and 55℃ for 10 s, and 72 ℃ for 2 min as the final extension following the manufacturer’s instructions (Mei5 Biotechnology, Co., Ltd). PCR amplicons were visualized on 1% agarose gels, purified and then subjected to bidirectional Sanger sequencing on an ABI 3730 XL instrument (Applied Biosystems, USA) using the same set of primers used for PCR amplification with BigDye v3.1 chemistry (Applied Biosystems) following manufacturer’s instructions.

Seven MLST loci (CAP59, GPD1, IGS1, LAC1, PLB1, SOD1, and URA5) were amplified and sequenced following the procedures of the International Society for Human and Animal Mycology (ISHAM) MLST consensus typing scheme for C. neoformans (http://mlst.mycologylab.org).^{25 (link)} Sequencing was performed using BigDye v3.1 Chemistry (Applied Biosystems, CA, USA) on an ABI 3130 Genetic Analyzer (Applied Biosystems, CA, USA). Both forward and reverse amplicons of each locus were sequenced. Consensus sequences were manually edited using ContigExpress and aligned in AlignX, implemented in VectorNTI Suite 7.0.^{26 (link)} A single Allele Type (AT) number was assigned to each of the seven loci by comparing consensus DNA sequences with the ISHAM database, resulting in a seven-number allelic profile for each isolate. The allelic profiles defined the corresponding STs.
MLST profiles and DNA sequences at each MLST loci for isolates from regions other than Laos and Vietnam were obtained from NCBI. For the global analysis, we used data reported by Simwami et al.,^{21 (link)} Mihara et al.,^{14 (link)} and Cogliati et al.^{27 (link)} included in Khayhan et al.,^{17 (link)} and from Chen et al.,^{28 (link)} Ferreira-Paim et al.,^{29 (link)} Beale et al.,^{30 (link)} and Dou et al.^{31 (link)}

Peripheral blood collected from the study participants was used for genomic DNA preparation using the standard protocol [42 (link)]. The target regions (detailed in Additional file 1) were amplified via polymerase chain reaction using primers (provided in Additional file 2) designed in the lab using the Primer3 software [43 (link)]. Applied Biosystems 3130 Genetic Analyzer with 98.5 % base calling accuracy and Read Length of upto 950 bp was used for sequence analysis of the amplicons using Big Dye v 3.1 chemistry and Sequencing Analysis Software, v 5.2 (Additional file 2). Chromatograms were also analyzed manually and mis-spaced letters/double peaks were investigated carefully for genotyping. For identification of heterozygous SNPs, >25 % base calling was accepted. Function of polymorphic variants was analyzed in silico using the is-rSNP [44 (link)].

The cDNAs encoding FLAG-aNSa1 (DU54816), FLAG-VHL-aNSa1 (DU54843), FLAG-aCS3 (DU54817), FLAG-VHL-aCS3 (DU54844), FLAG-aASC (DU54821), FLAG-VHL-aASC (DU54832) and human VHL (DU54023) were designed from published protein sequences [16 (link),17 ] and cloned into pBABED-Puro vectors (Cell Biolabs, modified; Dundee-modified version of the original Cell Biolabs pBABE plasmid) for constitutive expression. The retroviral expression system vectors pCMV-Gag-Pol and pCMV-VSVG constructs were from Clontech. pCDNA5 FRT/TO-HA-BCR-Abl (DU26928) was transiently expressed in cells where indicated in the figure legends. All DNA constructs were verified by DNA sequencing, performed by the DNA Sequencing and Services (MRCPPU, College of Life Sciences, University of Dundee, Scotland, http://www.dnaseq.co.uk) using Applied Biosystems Big-Dye v. 3.1 chemistry on an Applied Biosystems model 3730 automated capillary DNA sequencer. All constructs are available to request from the MRC-PPU reagents webpage (http://mrcppureagents.dundee.ac.uk) and the unique identifier (DU) numbers indicated above provide direct links to the cloning and sequence details. The pRetroX-Tet-On and pRetroX-Tight vectors for inducible AdPROM expression were obtained from Clontech. The non-targeting control siRNAs (cat.: D-001810-01-05) and the SHP2 (PTPN11) siRNA pool (cat.: L-003947-00-0005) were from Dharmacon.

Where possible, animals found to be homozygous at each of the five alleles identified in study B were selected. In total, 28 homozygotes were identified consisting of 18 allele 1, five allele 2, four allele 3 and one allele 4 animals, due to an absence of allele 5 homozygotes two heterozygotes were included. Skin gDNA was extracted as for genotyping; gDNA from lower genital tract tissue was extracted with the inclusion of an incubation step with alpha amylase (10% by volume) for 2 h at 37°C, prior to the addition of RNase A as per Buckles et al. [7 (link)]. A nested PCR protocol was used and PCR products of appropriate size (approx. 719 bp) were purified using MSB Spin PCRapace PCR purification kits (STRATEC Molecular, Germany). Where possible, 40 ng of DNA was submitted for sequencing. DNA sequencing was performed by DNA Sequencing & Services (MRCPPU, College of Life Sciences, University of Dundee, UK, www.dnaseq.co.uk) using Applied Biosystems Big-Dye v. 3.1 chemistry on an Applied Biosystems model 3730 automated capillary sequencer. The DNA sequences were analysed using the software program Geneious Pro v. 5.6.6 (Biomatters, available from http://www.geneious.com/).