Click
here to close Hello! We notice that
you are using Internet Explorer, which is not supported by Echinobase
and may cause the site to display incorrectly. We suggest using a
current version of Chrome,
FireFox,
or Safari.
Sci Rep
2015 Nov 23;5:16963. doi: 10.1038/srep16963.
Show Gene links
Show Anatomy links
MIG-seq: an effective PCR-based method for genome-wide single-nucleotide polymorphism genotyping using the next-generation sequencing platform.
Suyama Y
,
Matsuki Y
.
???displayArticle.abstract???
Restriction-enzyme (RE)-based next-generation sequencing methods have revolutionized marker-assisted genetic studies; however, the use of REs has limited their widespread adoption, especially in field samples with low-quality DNA and/or small quantities of DNA. Here, we developed a PCR-based procedure to construct reduced representation libraries without RE digestion steps, representing de novo single-nucleotide polymorphism discovery, and its genotyping using next-generation sequencing. Using multiplexed inter-simple sequence repeat (ISSR) primers, thousands of genome-wide regions were amplified effectively from a wide variety of genomes, without prior genetic information. We demonstrated: 1) Mendelian gametic segregation of the discovered variants; 2) reproducibility of genotyping by checking its applicability for individual identification; and 3) applicability in a wide variety of species by checking standard population genetic analysis. This approach, called multiplexed ISSR genotyping by sequencing, should be applicable to many marker-assisted genetic studies with a wide range of DNA qualities and quantities.
Figure 1. Construction of the MIG-seq library.(a) Multiple non-repetitive regions from various inter-simple sequence repeats (ISSRs) are amplified from genomic DNA by multiplexed PCR with tailed ISSR primers (1st PCR). (b) The 1st PCR products are subsequently used as the templates for the 2nd PCR (tailed PCR). This step enables the addition of complementary sequences for the binding sites of Illumina sequencing flow cell and indices (barcodes) for each sample to the 1st PCR products, using common forward and indexed reverse primers. (c) After measuring the approximate concentration of each 2nd PCR product, they are pooled in equimolar concentrations as a single mixture library. The mixture is then purified, fragments with a size range of 300–800 bp are isolated, the final concentration is measured by quantitative PCR, and is then used for Illumina paired-end sequencing (reads 1 and 2) and index reading. Sequencing of the first 17 nucleotides (primer region) of read 1, and 3 nucleotides (anchor region) of read 2 are skipped using the ‘DarkCycle’ option of the sequencer (indicated as the gray region in the arrows). (d) The sequence of the resulting library consists of binding sites for the P5 flow cell oligonucleotides and read 1 sequencing primer, forward ISSR primer, DNA insert, reverse ISSR primer, binding sites for read 2 and index sequencing primers, and P7 flow cell oligonucleotides.
Figure 2. An example of clone identification by MIG-seq analysis.(a) Spatial distribution of 18 ramets (samples) and three clonal groups (genets, enclosed by dotted line) of Sasa palmata in the Kawatabi Field Science Center, Tohoku University (the main building is indicated on the map), inferred from 144 SNP markers discovered by MIG-seq analysis. Circles indicate the sampling site of each ramet in the distribution area (dark gray) of the species. White and light-gray areas indicate cultivated fields and conifer plantations, respectively. White and blue circles indicate ramets in an inferred clone and ramets from different clones, respectively. The map was generated by YM using Adobe Illustrator CC 2015 version 19.1.0 (Adobe Systems, San Francisco, CA, USA). (b) Matrix of the differences between pairwise numbers of SNPs in the 144 SNP markers among 18 samples of the species. White and blue columns indicate pairs of inferred clones with a small number of different SNPs (0–6) and ramets from different clones with large numbers of different SNPs (27–71), respectively. (c) Histogram showing the frequency distribution of the differences between the pairwise numbers of SNPs in the 144 SNP markers among 18 samples of the species. White and blue bars show the first peak represented by pairs of inferred clones, and the second peak is represented by pairs of ramets from different clones, respectively.
Figure 3. Examples of population genetic analysis based on MIG-seq for eight samples from two different populations (sources) each of six species: (a) nameko mushroom (Pholiota microspora); (b) Calanoida copepod (Eodiaptomus japonicus); (c) Japanese common sea cucumber (Apostichopus japonicus); (d) predatory sea snail (Laguncula pulchella); (e) Carolina anole (Anolis carolinensis); (f) lady’s-slipper orchid (Cypripedium macranthos var. rebunense). Sampling locations (left), plots of principal coordinate analysis (center), estimation of the optimum number of clusters (K, right), and proportion of the membership coefficient of eight samples in the STRUCTURE analysis (upper right) are shown. (g) Their sampling areas on a large-scale map are also shown. The map was based on the blank map available from Geospatial Information Authority of Japan and modified by YM using Adobe Illustrator CC 2015 version 19.1.0 (Adobe Systems). Photos by Manami Kanno (a,c), Wataru Makino (b), Jotaro Urabe (d), and Yoshihisa Suyama (e,f).
Altshuler,
An SNP map of the human genome generated by reduced representation shotgun sequencing.
2000, Pubmed
Altshuler,
An SNP map of the human genome generated by reduced representation shotgun sequencing.
2000,
Pubmed
Andolfatto,
Multiplexed shotgun genotyping for rapid and efficient genetic mapping.
2011,
Pubmed
Baird,
Rapid SNP discovery and genetic mapping using sequenced RAD markers.
2008,
Pubmed
Barrett,
Haploview: analysis and visualization of LD and haplotype maps.
2005,
Pubmed
Botstein,
Construction of a genetic linkage map in man using restriction fragment length polymorphisms.
1980,
Pubmed
Catchen,
Stacks: building and genotyping Loci de novo from short-read sequences.
2011,
Pubmed
Davey,
Genome-wide genetic marker discovery and genotyping using next-generation sequencing.
2011,
Pubmed
Elshire,
A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species.
2011,
Pubmed
Gupta,
Amplification of DNA markers from evolutionarily diverse genomes using single primers of simple-sequence repeats.
1994,
Pubmed
Harismendy,
Detection of low prevalence somatic mutations in solid tumors with ultra-deep targeted sequencing.
2011,
Pubmed
Jakobsson,
CLUMPP: a cluster matching and permutation program for dealing with label switching and multimodality in analysis of population structure.
2007,
Pubmed
Kitamura,
Development of microsatellite markers for the dwarf bamboo species Sasa cernua and Sasa kurilensis (Poaceae) in northern Japan.
2009,
Pubmed
Lassmann,
TagDust--a program to eliminate artifacts from next generation sequencing data.
2009,
Pubmed
Miller,
Rapid and cost-effective polymorphism identification and genotyping using restriction site associated DNA (RAD) markers.
2007,
Pubmed
Morgante,
PCR-amplified microsatellites as markers in plant genetics.
1993,
Pubmed
Peterson,
Double digest RADseq: an inexpensive method for de novo SNP discovery and genotyping in model and non-model species.
2012,
Pubmed
Poland,
Development of high-density genetic maps for barley and wheat using a novel two-enzyme genotyping-by-sequencing approach.
2012,
Pubmed
Pritchard,
Inference of population structure using multilocus genotype data.
2000,
Pubmed
Stolle,
RESTseq--efficient benchtop population genomics with RESTriction Fragment SEQuencing.
2013,
Pubmed
Van Tassell,
SNP discovery and allele frequency estimation by deep sequencing of reduced representation libraries.
2008,
Pubmed
Vos,
AFLP: a new technique for DNA fingerprinting.
1995,
Pubmed
Wang,
2b-RAD: a simple and flexible method for genome-wide genotyping.
2012,
Pubmed
Zietkiewicz,
Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification.
1994,
Pubmed
van Orsouw,
Complexity reduction of polymorphic sequences (CRoPS): a novel approach for large-scale polymorphism discovery in complex genomes.
2007,
Pubmed