Next generation sequencing (NGS) is changing the way to analyse and extract genetic information from all species. One of the most promising NGS platforms is represented by the Ion Torrent PGM (Personal Genome Machine) technology. The sequencing process of this machine is based on the detection of pH variation which occurs when H+ is released during a nucleotide incorporation in the sequencing reaction. This chemical signal is then converted directly to a digital information. In this study we applied the Ion Torrent PGM technology to identify single nucleotide polymorphisms (SNPs) in the rabbit genome, a species for which massive SNP information is not available yet. Two reduced representation libraries (RRLs) were prepared. Genomic DNA pools were constructed with equimolar DNA of 10 rabbits from 4 breeds (Burgundy Fawn, Champagne d’Argent, Checkered Giant and Rhinelander) and from a commercial line. Pooled DNA was digested separately with two different restriction enzymes (HaeIII) and (RsaI). Digested DNA was electrophoresed on agarose gels from which a slice containing DNA fragments of about 500-600 bp was cut out and used to extract DNA. The isolated DNA was then prepared for the sequencing on 318 Ion Torrent chips following manufacturer instructions. From the two RRLs a total of 697.79 Mb (609.38 Mb with base quality > 20), derived from 6,964,750 reads (with a mean length of 100 bp) were sequenced. Of these reads, 6,312,660 were mapped on the reference rabbit genome sequence (oryCun2.0) and then used for variant calling analysis. Retaining only single nucleotide variation with a mapping quality >10 and detected in at least 4 bases, 65,695 SNPs were identified, with a mean distribution of 1 SNP every 287 bp. Annotation of these SNPs was based on the oryCun2.0 genome version. Some of the putative SNPs were validated by visual inspection using IGV (Integrative Genomics Viewer) and by Sanger sequencing. These SNPs could be useful to design a commercial SNP genotyping platform for the rabbit.
Application of the Ion Torrent technology to identify single nucleotide polymorphisms in the rabbit genome
BERTOLINI, FRANCESCA;SCHIAVO, GIUSEPPINA;SCOTTI, EMILIO;RIBANI, ANISA;MARTELLI, PIER LUIGI;CASADIO, RITA;FONTANESI, LUCA
2013
Abstract
Next generation sequencing (NGS) is changing the way to analyse and extract genetic information from all species. One of the most promising NGS platforms is represented by the Ion Torrent PGM (Personal Genome Machine) technology. The sequencing process of this machine is based on the detection of pH variation which occurs when H+ is released during a nucleotide incorporation in the sequencing reaction. This chemical signal is then converted directly to a digital information. In this study we applied the Ion Torrent PGM technology to identify single nucleotide polymorphisms (SNPs) in the rabbit genome, a species for which massive SNP information is not available yet. Two reduced representation libraries (RRLs) were prepared. Genomic DNA pools were constructed with equimolar DNA of 10 rabbits from 4 breeds (Burgundy Fawn, Champagne d’Argent, Checkered Giant and Rhinelander) and from a commercial line. Pooled DNA was digested separately with two different restriction enzymes (HaeIII) and (RsaI). Digested DNA was electrophoresed on agarose gels from which a slice containing DNA fragments of about 500-600 bp was cut out and used to extract DNA. The isolated DNA was then prepared for the sequencing on 318 Ion Torrent chips following manufacturer instructions. From the two RRLs a total of 697.79 Mb (609.38 Mb with base quality > 20), derived from 6,964,750 reads (with a mean length of 100 bp) were sequenced. Of these reads, 6,312,660 were mapped on the reference rabbit genome sequence (oryCun2.0) and then used for variant calling analysis. Retaining only single nucleotide variation with a mapping quality >10 and detected in at least 4 bases, 65,695 SNPs were identified, with a mean distribution of 1 SNP every 287 bp. Annotation of these SNPs was based on the oryCun2.0 genome version. Some of the putative SNPs were validated by visual inspection using IGV (Integrative Genomics Viewer) and by Sanger sequencing. These SNPs could be useful to design a commercial SNP genotyping platform for the rabbit.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
