So far we’ve been doing microsatellite analyses with a capillary sequencer. The machine is getting old and we need to replace it. Since we’re regularly using MiSeq or HiSeq for other approaches (DNA metabarcoding, Rad-seq, Genotyping By sequencing, …), it seems logical to us to switch completely to a high throughput sequencing platform.
We do work a lot on protected and endangered species, often small critters, as a result of which our DNA-sampling needs to be non-lethal and minimally invasive. This means we often have low quantity and low quality DNA.
RAD-sequencing or other reduced representation genome sequencing is often not possible on these types of samples. I am aware of SNP-methods requiring low amounts of DNA (Fluidigm chips for example), but this still requires other hardware.
Microsatellite genotyping is still very useful to us because the information content per locus is about ten times higher than with SNPs. For a lot of our purposes, we don’t need more than 25 microsatellite loci to get the information we need, and with the small amounts of DNA we have at our disposal this works fine.
I’m looking for people who directly sequence microsat loci via high throughput sequencing, and would like to know their first-hand experiences on feasibility, bottlenecks, bio-informatic pipelines, do’s and don’ts.
thanks
I'm interested in this topic too, so I would be interested in what you find. Couple of thoughts: I agree that microsatellites have more information content than SNPs, but I think SNPs have some advantages. Easier to genotype, can use a smaller fragment length, and a lot more can be genotyped at one time. I have thought about amplicon sequencing with high-throughput sequencers. Check out this paper where several thousand samples can be genotyped at once at a couple hundred of SNPs ( Article Genotyping-in-Thousands by sequencing (GT-seq): A cost effec...
). Our lab has used this technique and I've talked to the authors about getting it to work for non-invasive samples. Could be an option. They have SNP panels that only need a 75 cycle flow cell to capture that actual SNP in each locus.Another lab we work with is using a similar approach except they are genotyping multiple SNPs per locus. They call them microhaplotypes and it delivers much more statistical power because there is greater information content per locus. Check it out (
https://doi.org/10.1111/1755-0998.12737).
I am curious how other people are doing with microsatellite genotyping.
Hello,
yes, you can do genotyping with Miseq, as you can see here. Article High-throughput microsatellite genotyping in ecology: Improv...
Also read about the use of Illumina in forensics (Illumina offical website), I found it very useful to see what can be done with low quality human DNA, I guess it should work with any DNA and the rigth PCR primers.
Best, Meta
I am also interested in the topic. Big manufacturers (Promega, AB) are still selling (and new versions of) the classical CE platforms. Beware of 'microhaplotypes' - lots of theretical and statistical problems.
I found some additional answers that might be of interest to all of you.
First, MipSTR is a NGS method for sequencing microsats. A link to the principle and the reference to the paper:
http://enseqlopedia.com/wiki-entry/dna-sequencing-methods/low-level-dna-detection/mipstr/ https://www.biorxiv.org/content/biorxiv/early/2014/07/28/007500.full.pdf
Next, there's an interesting paper by Chiou & Bergey 2018 on targeted enrichment of vertebrate DNA from mixed DNA samples such as faeces. This might help to overcome issues in GBS and radseq from some types of samples
SCiENtifiC REPORTS | (2018) 8:1975 | DOI:10.1038/s41598-018-20427-9
And another paper (Suyama & Matsuki 2015) on SNP sequencing from reduced representation samples that is based on STR-repeat recognition to construct a reduced representation library. Instead of requiring large amounts of DNA for restriction and ligation of DNA, it uses STR-repeats and PCR to amplify a reduced portion of the genome and then sequence this.
https://www.researchgate.net/publication/284565206_MIG-seq_An_effective_PCR-based_method_for_genome-wide_single-nucleotide_polymorphism_genotyping_using_the_next-generation_sequencing_platform
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