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[…] sing 1°C each cycle until 55°C) and 15 s at 72°C; and a final melting step of 1 min at 95°C, 1 min at 40°C, and 1 s at 60°C with the temperature being raised by 0.02°C/s until 95°C was reached., GBS was performed in 83 plants comprising 73 pre-selected plants from different generations (BC5S1, BC4S1, and BC2S1), 2 samples of each parent, 2 additional samples of doubled haploids of the recurrent parent and 2 samples of the hybrid. After DNA extraction and dilution, the samples were sent to the Cornell University genomic facilities in August 2015 for library preparation (a PstI enzyme was used) and sequencing. The raw data were demultiplexed and the Illumina barcode removed using the software GBS Barcode Splitter (https://sourceforge.net/projects/gbsbarcode/). Subsequently, the sequences were filtered at a 25 Phred quality score and the Illumina adapters removed with the Fastq-mcf program (Aronesty, ). A quality control before and after the trimming was performed with the FastQC tool (Andrews, ) and general statistics were obtained using Fastq-stats (Aronesty, ). Then, the clean reads were mapped against the reference published genome (Hirakawa et al., ) and against another eggplant genome with improved quality, kindly provided by the Italian Eggplant Genome Sequencing Consortium (Barchi et al., ). Due to the better assembly of the latter (12 pseudomolecules vs. 33,873 scaffolds of the former), we decided to use it for the further analysis. The mapping was performed using BWA-MEM software (Li, ) with default settings and the SNP calling using the FreeBayes program (Garrison and Marth, ), ignoring those variants that presented less coverage than 15, a mapping quality of 20 or a base quality of 20. In order to select the most reliable SNPs for genotyping the plants with HRM, different filters from the Seq Crumbs tools (https://bioinf.comav.upv.es/seq_crumbs/) were applied to the VCF file for maximizing the polymorphism validation. The filters CS60 and CL60 identified polymorphisms that were at a distance of < 60 nucleotides to another polymorphism or to the alignment edge, respectively. The filter HV0.05 determined whether the region had more than 5 polymorp […]

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