EasySeq™ NGS Targeted Capture Kits for Genotyping by NGS

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A proven method for High-Throughput Micro-Haplotyping

  • Cost effective
  • High throughput
  • High multiplexing
  • Easy workflow

 

Abstract
Marker assisted breeding (MAB) is an indirect selection process where a trait is ­selected, based on a linked marker to the trait of interest (e.g. productivity, disease ­resistance, ­quality). Currently, this application is often supported by monoplex Genotyping ­assays, such as TaqMan or KASP. Current breeding programs are studying more and more ­complex traits, introducing the need for more high-throughput, multiplex and cost ­effective Genotyping methods. Microarrays have been implemented in some cases, but have the drawback of low flexibility and high startup costs.

With the strength of Genotyping by NGS and the extremely user friendly workflow and flexibility, the EasySeq NGS target capture kits represent a method that combines ­flexibility, high to ultra high thoughput in a multiplex SNP Genotyping method that also enables micro haplotyping, utilizing tailor-made Molecular Inversion Probes (smMIPs). The application of the EasySeq™ NGS Targeted Capture kit is delivering new ­opportunities for HZPC in identifying chromosomal loci that play a role in complex potato traits (such as starch content, sugar content, and pest ­resistance).

 Potatos

The improved lab efficiency from the workflow and the increased throughput ­capabilities fit the needs of the company and make the EasySeq™ NGS Targeted ­Capture kits an ideal cost effective solution.

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Smmip Overview And Schematic Workflow 11Panel-design
Using MIPgen software the target smMIP-­probes are designed based on the ­reference DM potato genome. Each smMIP-probe is ­covering a genomic segment of 120 bp, ­containing at least one SNP of interest. One run of the MIPgen software simultaneously designs all smMIP-­probes requested in the BED-file, and these multiple targets will be combined in one EasySeq™ NGS Targeted Capture assay. In the current assays the number of combined ­smMIP-probes is 71, this can easily be ­expanded to approximately 2000 smMIP-probes. Each smMIP-probe harbors a unique molecular identifier (UMI), that facilitates ­removal of PCR duplicates later on in the data analysis.

smMIP synthesis
The created smMIP-probe sequences are ­synthesized by Biolegio (Nijmegen, The Netherlands) for synthesis, 5’phosphorylation is done during synthesis. Having the smMIPs phosphorylated during synthesis improves performance by diminishing self-ligated smMIPs in the final PCR product.

Wet-lab:
Delivered smMIP probes are pooled equimolar, to facilitate the combined use in one multiplex assay. The target read depth is 400 X on ­average for each fragment smMIP The EasySeq™ NGS Targeted Capture is ­performed in a 384 well plate, according to the protocol developed in cooperation with HZPC. To increase the possibilities for sample pooling, both the P5 and P7 amplification primers are equipped with an 8bp index (768 combinations available, 48X i7 plus 16x i5). Validation of successful product formation (smMIP + region of interest) is ­tested in selected random samples is verified with the Agilent BioAnalyzer using a DNA1000 chip. Subsequently, EasySeq™ NGS Targeted Capture libraries derived from up to 768 ­samples were pooled and the pool was purified with a Qiagen® PCR purification kit in order to ­exchange buffers and adapter-primers.

Figure Overall Sequencing MetricsTypical overall sequencing metrics show 92% Pass Filter, 91% Q30 and 0.3% error rate. This means that the libraries can be sequenced with a high degree of confidence on the ­MiniSeq®. Microhaplotypes and allele dosages of all ­target fragments could be extracted from the data ­after removal of PCR duplicates using the ­smMIP single molecule tag / UMI. For an even coverage between the smMIPs the pool was rebalanced twice on test samples ­before running the true experiments

Microhaplotype
Next to the known SNPs for which the smMIP was designed. Novel SNPs were identified in the HZPC bioinformatics pipeline and these will then be used in the microhaplotype

Application note
Poster

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