Application of Sage Science Nucleic Acid Fragment Recovery System in RADseq

1. What is RAD-SEQ?
RADseq (Restriction-site associated DNA sequencing) is a low-cost process by cleavage of the genome and sequencing of DNA-related fragments with restriction sites. A large number of SNP markers were developed, independent of the presence or absence of reference genomes and chromosomal ploidy.
2. Where is its application?
RADseq technology has been widely used in population evolution studies of animal and plant, genetic map construction and QTL mapping, and genome-wide association analysis. Its dominant application is the SNP search and genotyping study in the ecological and evolutionary studies of non-model species .
3. What kind of RAD-seq?
1) RestSeq (restriction fragment sequencing): restriction enzyme I digestion + ligation linker + restriction enzyme II digestion + fragment selection database construction;
2) ezRAD: one or more common enzyme digestion + illuminate database build library sequencing;
3) SLAF-seq (specific-locus amplified fragment sequencing): enzyme I digestion + barcode + enzyme II digestion + linker library sequencing;
4) 2bRAD: IIB restriction enzyme single digestion, type IIB restriction enzymes can be cut at the upstream and downstream sites of the recognition site, respectively, to obtain a fixed length fragment. Constituting a 33–36 bp insert for library sequencing;
5) ddRAD (double-digest restriction-site-associated DNA): two restriction enzymes were digested, and the linker was matched with an enzyme + gel to carry out fragment selection and library sequencing;
6) SBG (sequence-based genotyping): rare enzyme and one or two common enzyme digestion + PCR selective amplification of short fragment sequences for library sequencing;
7) MSG (multiplexed shotgun genotyping): common enzyme single enzyme digestion + fragment selection database construction;
8) GBS (genotyping by sequencing): common enzyme single enzyme digestion + PCR selective amplification of short fragment sequences for library sequencing;
9) RRL (reduced representation library): the restriction endonuclease digestion + fragment selection database construction;
10) original RAD (original restriction-site associated-DNA): restriction enzyme single enzyme digestion + mechanical interrupting database construction;
11) CRoPS (complexity reduction of polymorphic sequences): two enzyme digestion + AFLP product library sequencing;

4. What is the significance of DNA fragment size screening during RAD-seq library construction?
Enzymatic digestion can simplify the genome into many DNA fragments. The fragment size screening step can further reduce the number of loci that need to be genotyped. Screening for potential characteristic gene loci depends primarily on the distance between the two restriction sites. The consistency of fragment screening between different sequencing libraries is crucial for generating comparable gene loci between different samples, which directly affects subsequent bioinformatics analysis and application. There are direct screening and indirect screening for fragment screening. Direct screening includes manual cutting, magnetic bead screening and pippin, but different techniques have different precision. The figure below is a simple comparison of different technical means.
Manual cutting is based on the leftmost marker control cut, even for the same person, it is difficult to guarantee the consistency of each lane and the scope of each sample.
The range of fragments purified by magnetic beads is too broad to focus.
Compared to manual and magnetic bead purification, pippin automated fragment recovery is more consistent and accurate, with repeatability and accuracy of over 95% . In the construction of ezRAD and ddRAD libraries, the consistency of pippin fragment recovery is a key factor in the success of the experiment. Dr. Brant Peterson , founder of ddRAD , believes that “even in the best experimental case of manual gel cutting, after the analysis of the biometric analysis, The effect of hand-cutting is only half that of the pippin instrument." Since the release of Pippin Prep, the first generation product in 2011, Blue Pippin, SageELF, PippinHT, SageHLS and other models have been launched. Sage science has become a leading brand in the field of nucleic acid fragment recycling.
Huanya Biotech Co., Ltd. is the general agent of Sage Company in China. It has been paying attention to the introduction and development of sequencing related equipment in China. It is committed to assist domestic customers to complete the preparation of sequencing samples better and faster, and achieve higher efficiency. Easier workflow.
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references:
Andrews KR, Good JM, Miller MR, et al. Harnessing thepower of RADseq for ecological and evolutionary genomics. [J]. Nature ReviewsGenetics, 2016
Peterson, BK, Weber, JN, Kay, EH, Fisher, et al. Double digest RADseq: an inexpensive method for de novo SNP discovery and genotyping in model and non-model species. [J]. PLOS ONE , 2012

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