Abi 3130xl automated sequencer

Genomic DNA was isolated from fetal skin sample using the QIAamp DNA Blood Midi kit (QIAGEN GmbH, Hilden, Germany) following the manufacturer’s recommendations. The coding sequences and intron-exon junctions of ESCO2 (GenBank NM_001017420.2) were directly sequenced. Purified PCR amplification products were sequenced using the BigDye Terminator Cycle Sequencing Kit v.1.1 (Applied Biosystems, Foster City, California, USA) according to the manufacturer’s instructions, and were resolved on an ABI 3130xl automated sequencer (Applied Biosystems). Sequence data were aligned with SeqScape 2.0 software and compared to the published sequences of ESCO2.

Nine neonates with E. meningosepticum meningitis who presented to the Neonatal Intensive Care Unit of Niloufer Hospital for Women and Children, Hyderabad, India, between January 2009 and December 2010, were included in the study. Cerebrospinal fluid collected from the neonates was subjected to direct microscopic examination, inoculated on to blood agar, chocolate agar, and MacConkey's agar, and incubated overnight at 37°C. Blood cultures were done by automated blood culture systems (Bact/Alert, Biomerieux, France). The isolates were identified by conventional biochemical reactions and Vitek 2 (Biomerieux, France). The identity of the isolates was further confirmed by 16S rRNA gene sequencing. Sequencing was performed (forward primer, 5′-TTGGAGAGTTTGATCCTGGCTC-3′; reverse primer, 5′-GGACTACCAGGGTATCTAA-3′) with fluorescence-labeled dideoxynucleotide terminators using an ABI 3130 Xl automated sequencer, following the manufacturer's instructions (PE Applied Biosystems). The sequences were analysed and identified using the Megablast search program of the GenBank database. The sequence of the isolate perfectly (100%) matched the sequences of E. meningosepticum deposited in GenBank. The gene sequences of the isolates were deposited in the GenBank. Antibiotic susceptibility of the isolates was done by Kirby Bauer's disk diffusion method and also by Vitek 2.

We selected a panel of 39 canine autosomal microsatellites (seven tetranucleotides and 32 dinucleotides) that were used in some of the most recent studies on wolf population genetics and hybridization in Europe ([24 ] and references therein). These microsatellites mapped on 26 different chromosomes (S1 Table) and were not in linkage disequilibrium in the studied populations. The panel includes 15 markers from the Finnzymes Canine Genotypes^™ Panel 1.1 multiplex kit (Finnzymes, Thermo Fisher Scientific, Vantaa, Finland). One of them, the Amelogenin, was used to sex the individuals. The microsatellites were amplified in eight PCR multiplexes using the Qiagen Multiplex PCR Kit (Qiagen, GmbH-Hilden, Germany). Negative (no DNA) and positive (samples with known genotypes) PCR controls were used to check for laboratory contaminations. To confirm allele calls, all samples were independently analysed twice to check for the occurrence of allelic dropout and false alleles, which were never observed. The amplicons were analysed in an ABI 3130XL automated sequencer (Applied Biosystems; Foster City, California, USA) and allele sizes were estimated using the software Genemapper 4.0. Details on the selected markers, primers and PCR profiles are available in the S1A Appendix.

All the variants of class 3 (variants uncertain significance) class 4/5 (likely pathogenic/pathogenic) and the novel variants detected by NGS were confirmed by bidirectional Sanger sequencing.
The sequencing was performed using the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) and the ABI 3130xl Automated Sequencer (Applied Biosystems, Foster City, CA, USA). The results were analyzed using Sequencing Analysis 5.2.0 software.

The coding and regulatory parts of ompF and ompC genes were amplified using OmpF/C-Bam_for and OmpF/C-Hind_rev primers and sequenced with internal primers (Table S3) to cover all regions. DNA sequencing was done with an ABI 3130 xl automated sequencer (Applied Biosystems, Waltham, MA, USA) and the ABI Prism dye terminator sequencing kit (Applied Biosystems, Waltham, MA, USA) according to the manufacturer’s directions.

Mutational profile by direct DNA sequencing of CXCR4 was carried out as previously described by our group [63 (link)]. In detail, PCR products were purified and sequenced from both sides using the BigDye terminator chemistry 3.1 (Applied Biosystems, Foster City, CA, USA). Sequences were run on an ABI3130-xl automated sequencer (Applied Biosystems, Foster City, CA, USA). For data analysis, the genebank file for CXCR4 (NG_011587.1) was used. The nucleotide acid sequences for the primer for these purposes are shown in Supplementary Table S2.

Genomic DNA was extracted from a small fraction of tissue from the posterior part of juveniles as well as from fin clips of the adults, and genotyped with six polymorphic microsatellite loci – UNH2101 (Stewart and Albertson 2010), Abur25, Abur44, Abur61, Abur98 and Abur117 (Sanetra et al. 2009) – which had been used in our previous population genetics study of P. microlepis (Lee et al. 2010). Forward primers were labeled with a fluorescent dye (6‐FAM or HEX). PCR reaction was carried out in 10 μL volumes (1× PCR buffer, 25 μmol/L of each dNTP, 0.5 μmol/L of each of the forward and reverse primers, 0.1 U Taq polymerase [Life Technologies, Darmstadt, Germany] and 30–50 ng of DNA). Some juvenile samples were failed to be successfully amplified at one or two loci (Table S1). PCR products were diluted in formamide HiDi and electrophoresed in an ABI 3130xl automated sequencer (Applied Biosystems, Darmstadt, Germany). Fragment sizes were compared to ROX 500 bp size standard (Applied Biosystems) as determined using GeneMapper software 4.0 (Applied Biosystems).