Author(s): C. Calabuig | M.D.N. Rodrigues | C.G.A. Moreira | D.B. Almeida | M. Katzenberger | A. Santos Júnior | G.B. Oliveira | H.L.M. Moreira
Journal: Genomics and Quantitative Genetics
ISSN 2157-9903
Volume: 5;
Issue: 1;
Start page: 14;
Date: 2012;
Original page
Keywords: amplifiable locus | Brazil | genetic diversity | migratory fowl
ABSTRACT
Microsatellites for coscoroba swan (Coscoroba coscoroba) were developed using Next Generation Sequencing (NGS) without prior enrichment. A shotgun/paired-end library was prepared according to standard protocol of Illumina Nextera DNA Library Kit with dual indexing. The PAL_FINDER_v0.02.03 Perl script was used to extract readings containing microsatellites with di-, tri-, tetra-, penta- and hexanucleotides from five million readings obtained in sequencing. Readings were grouped and used in Primer3 version 2.0.0 to design primers when GC content greater than 30%, melting temperatures within 58-65 oC, with 2 oC maximum difference between primers, the last 2 nucleotides in 3’ extremity are G or C, and maximum poli-N of 4 nucleotides. When all criteria were met, a single pair of primers was selected according to the highest score in Primer3 and with greater amplification region of the repeated sequence. We identified 3046 potentially amplifiable loci (microsatellites) of which 52 were hexanucleotides, 447 pentanucleotides, 760 tetranucleotides, 1168 trinucleotides and 618 dinucleotides. The most common microsatellite motifs in each size were: TC (51.6%) for dinucleotides; ATT (23.9%) for trinucleotides; AAAT (31.4%) for tetranucleotide; ATAGG (60.6%) for pentanucleotides; and AAAAAG (21.2%) for hexanucleotides. A group of ten loci (including di-, tri-, and tetranucleotides) has been sequenced, using Sanger’s method, to obtain complete sequences for fragments over 200 bp and are currently being used to study the genetic diversity of coscoroba swan populations. Microsatellites acquired with NGS are an efficient tool for obtaining highly polymorphic markers for non-model species.
Journal: Genomics and Quantitative Genetics
ISSN 2157-9903
Volume: 5;
Issue: 1;
Start page: 14;
Date: 2012;
Original page
Keywords: amplifiable locus | Brazil | genetic diversity | migratory fowl
ABSTRACT
Microsatellites for coscoroba swan (Coscoroba coscoroba) were developed using Next Generation Sequencing (NGS) without prior enrichment. A shotgun/paired-end library was prepared according to standard protocol of Illumina Nextera DNA Library Kit with dual indexing. The PAL_FINDER_v0.02.03 Perl script was used to extract readings containing microsatellites with di-, tri-, tetra-, penta- and hexanucleotides from five million readings obtained in sequencing. Readings were grouped and used in Primer3 version 2.0.0 to design primers when GC content greater than 30%, melting temperatures within 58-65 oC, with 2 oC maximum difference between primers, the last 2 nucleotides in 3’ extremity are G or C, and maximum poli-N of 4 nucleotides. When all criteria were met, a single pair of primers was selected according to the highest score in Primer3 and with greater amplification region of the repeated sequence. We identified 3046 potentially amplifiable loci (microsatellites) of which 52 were hexanucleotides, 447 pentanucleotides, 760 tetranucleotides, 1168 trinucleotides and 618 dinucleotides. The most common microsatellite motifs in each size were: TC (51.6%) for dinucleotides; ATT (23.9%) for trinucleotides; AAAT (31.4%) for tetranucleotide; ATAGG (60.6%) for pentanucleotides; and AAAAAG (21.2%) for hexanucleotides. A group of ten loci (including di-, tri-, and tetranucleotides) has been sequenced, using Sanger’s method, to obtain complete sequences for fragments over 200 bp and are currently being used to study the genetic diversity of coscoroba swan populations. Microsatellites acquired with NGS are an efficient tool for obtaining highly polymorphic markers for non-model species.
