3500 genetic analyzer

Mutational analysis of RET gene was carried out by using standard PCR and Sanger sequencing protocols. Briefly, peripheral blood from MTC patients was collected and DNA was automatically extracted by using MagNA Pure 2.0 (Roche Diagnostics®, Switzerland) platform. PCR was performed with a High Fidelity PCR kit (Roche Diagnostics) following protocol according to Jindřichová et al. [10 (link)]: initial denaturation at 95°C for 10 min followed by 40 cycles (of denaturation at 95°C for 30 s, annealing at optimized temperature (60–68°C) for 30 sec, and elongation at 72°C for 1 min) and final elongation at 72°C for 10 min. The PCR products were evaluated with a Qiaxell system (Qiagen Company®, Hilden, Germany) and mutations were assessed through Sanger sequencing in a 3500 Genetic Analyzer (Life Technologies®, California, United States) by using BigDye 3.1 chemistry.

Sanger sequencing was performed on the same amplicons as used for HRM analysis. 5 μl of PCR products were purified with exonuclease I and Fast-AP (Thermo Fisher Scientific, Waltham, USA) for 15 min at 37°C and 15 min by 80°C. A sequencing reaction was set up with 1 μl of purified PCR products and the BigDye® Terminator v1.1 Cycle Sequencing Kit (Life Technologies) following the manufacturer’s instructions. The BigDye XTerminator® Purification Kit (Life Technologies) was used for the purification of the DNA sequencing reactions removing non-incorporated BigDye® terminators and salts. Solution was incubated for 30 min with agitation of 1800 rpm. Sequencing analyses were carried out on the eight capillary 3500 Genetic Analyzer (Life Technologies).

A 3 primer PCR assay was developed for genotyping SNNL1, with a forward and reverse primer flanking the insertion site and 1 primer internal to the retrocopy. Internal primers were then used for sequencing the entire retrocopy. Primers were also designed for genotyping the SLC26A4 chr18:12,910,382 C/T variant. All primers were developed using Primer3 software (Untergasser et al. 2012 (link)). The primers used in this study are available in Supplementary Table 1. Sanger sequencing was performed on an Applied Biosystems 3500 Genetic Analyzer using a Big Dye Terminator Sequencing Kit (Life Technologies, Burlington, ON, Canada). Additional genotyping of SNNL1 from WGS data was performed visually in IGV. Samples which had no reads crossing either of the breakends at the insertion site were considered homozygous for the retrocopy insertion.

We resolved the retroCNV insertion sites using a previously developed pipeline [17 (link)]. Briefly, we aligned WGS fastq files to the EquCab3.0 reference assembly [35 (link)] using Minimap2 v2.24 with the preset ‘-ax sr’ for Illumina paired end reads [36 (link)]. Aligned data were sorted and duplicate reads were removed using samtools [37 (link)]. We then used TEBreak to obtain discordant read clusters at putative retroCNV parent gene loci [38 (link)]. We visually confirmed all retroCNV insertion sites using IGV [39 (link)]. The TEBreak 5’ and 3’ junction sequences for the retroCNVs are available in S2 Table. To validate a set of retroCNV insertion sites and TSD, we designed three primer genotyping PCR assays as previously described [26 (link)] (S3 Table). For genotyping, we randomly selected thoroughbred horses from a DNA repository maintained by the Bannasch lab. These DNA samples were collected. We performed sanger sequencing on an Applied Biosystems 3500 Genetic Analyzer using a Big Dye Terminator Sequencing Kit (Life Technologies, Burlington, ON, Canada). We also analyzed the horse Y chromosome assembly eMSYv3.1 (GenBank accession MH341179) [40 ] for evidence of retroCNVs that had been predicted to be on the Y chromosome based on sex bias. The retroCNV parent gene sequence was used to query the Y chromosome for the retrocopy using BLAST [41 ].

EmsB PCR amplification was performed as previously reported (17 (link)). Capillary electrophoresis of PCR products was performed on a 3500 genetic analyzer (Life Technologies, Foster City, CA, USA). The size and height of each peak of the electrophoretic profile constituting the EmsB profiles were determined with the use of GeneMapper 4.1. The characterization of EmsB profiles composed of several peaks or alleles from 209 to 241 bp was carried out as previously described (11 (link), 20 (link)). The hierarchical clustering analysis was done using the Euclidean distance and the average link clustering method (UPGMA) (22 ). The uncertainty of clusters was evaluated by multiscale bootstrap resampling (B = 1,000) and given by approximately unbiased p-values (AU), according to Shimodaira (23 (link), 24 ). Clustering analyses were performed using R statistical software (25 ) and the pvclust library (26 (link)). In each dendrogram, EmsB microsatellite data from previously genotyped samples from the Arctic and Asian groups (11 (link), 13 (link)) were added. The genetic threshold of 0.08 was used to determine the genotyping status of each sample (11 (link)), while two E. granulosus sensu stricto (G1) samples were used as the outgroup.

PCR products were purified using NucleoSpin Gel and PCR Clean-up kit (MACHEREY-NAGEL) as per manufacture’s protocol and quantified using Nano-Drop 2000c Spectrophotometer (Thermo Fisher Scientific Inc.) and submitted for sequencing in capillary electrophoresis 3500 Genetic Analyzer (Life Technologies). Sanger sequencing traces were analysed for mutation using Mutation Surveyor [38 (link)]. The details of all the primers used for mutation analysis have been provided in Additional file 2: Table S7.

Partial AZFc microdeletions were analyzed by plus/minus amplification of validated Sequence Tagged Sites (STS) markers located in the Y chromosome [9] (link), [16] (link). Briefly, markers sY142, sY1197, sY1191, sY1291, sY1206, sY1201 and sY1261 have been amplified. Amplification was performed in a multiplex reaction with 12.5 µL Multiplex PCR Master Mix (QIAGEN, Germany), 5 pmol FAM-labeled M13 primer (FAM-TGTAAAACGACGGCCAGT), unlabeled primers (listed in Table S1) and 2 µL template DNA (ranging 1–10 ng/µL) in a total volume of 25 µL. The cycling conditions were the following: 15 minutes for Taq polymerase activation at 95°C, 30 cycles of 95°C 30 sec, 60°C 30 sec and 72°C 30 sec, and 8 cycles of 95°C 30 sec, 53°C 30 sec and 72°C 30 sec, followed by 30 minute extension at 72°C. All PCR reactions were performed in a Verity Thermal Cycler (Life Technologies, USA). PCR primerś sequences have been designed with M13 tails for product detection with M13 primers labeled with FAM, as previously described [29] . PCR productś resolution and detection was performed with a 3500 Genetic Analyzer (Life Technologies, USA) and the analysis of the results was performed with GeneMapper ID-X v1.2 software (Life Technologies, USA).