Hi all,
I am new to NGS and was hoping to receive some advice regarding the experimental design for a project we are currently putting together in which we are looking to ID informative SNPs for detecting introgression between multiple species with a convoluted history of much hybridization/ reticulations. Genome size approx. 2.2 gb w/ estimate of 38% GC content. The “closest” genomic resource available is within family, different genera. Other (potentially pertinent?) details: We think that 20X coverage as a minimum would be appropriate, and are looking to multiplex at least 96 individuals per lane.
Question: Does it make sense to genotype all (1000+) of our samples using RAD (or ddRAD?), or should we use RADseq on a subset of individuals (96 individuals total from “pure” populations?), ID informative SNPs, and then screen the rest of our samples using some genotyping assay (e.g. Sequenom)? Many projects seem to go this direction, but the problem I see is that it requires that there be enough flanking sequence (to the SNP) to develop oligos for Sequenom.
We will be running this on an Illumina HiSeq 2000, which yields fragments that I think may be too small (~100 bp) for us to be able to develop flanking oligos for use with Sequenom, unless the SNP happens to be smack in the center of the fragment, right? Does anyone have any experience with the Sequenom MassArray platform for SNP genotyping and could shed light on this issue?
Alternatively, we may have access to a MiSeq to yield larger fragment sizes, or we could use an overlapping paired-end method to try and get longer fragments as well (Hohenlohe et al. 2013; Molecular Ecology).
Any help/ direction is much appreciated.
I am new to NGS and was hoping to receive some advice regarding the experimental design for a project we are currently putting together in which we are looking to ID informative SNPs for detecting introgression between multiple species with a convoluted history of much hybridization/ reticulations. Genome size approx. 2.2 gb w/ estimate of 38% GC content. The “closest” genomic resource available is within family, different genera. Other (potentially pertinent?) details: We think that 20X coverage as a minimum would be appropriate, and are looking to multiplex at least 96 individuals per lane.
Question: Does it make sense to genotype all (1000+) of our samples using RAD (or ddRAD?), or should we use RADseq on a subset of individuals (96 individuals total from “pure” populations?), ID informative SNPs, and then screen the rest of our samples using some genotyping assay (e.g. Sequenom)? Many projects seem to go this direction, but the problem I see is that it requires that there be enough flanking sequence (to the SNP) to develop oligos for Sequenom.
We will be running this on an Illumina HiSeq 2000, which yields fragments that I think may be too small (~100 bp) for us to be able to develop flanking oligos for use with Sequenom, unless the SNP happens to be smack in the center of the fragment, right? Does anyone have any experience with the Sequenom MassArray platform for SNP genotyping and could shed light on this issue?
Alternatively, we may have access to a MiSeq to yield larger fragment sizes, or we could use an overlapping paired-end method to try and get longer fragments as well (Hohenlohe et al. 2013; Molecular Ecology).
Any help/ direction is much appreciated.
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