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Allegro® Targeted Genotyping V2

Complete end-to-end solution for targeted genotyping-by-sequencing. Fast, scalable, cost-effective, with high sample multiplexing capability.




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Tab 01 / Overview
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Efficient and cost-effective SNP interrogation

Allegro Targeted Genotyping V2 provides a fast, scalable, cost-effective approach to perform targeted genotyping-by-sequencing on a wide variety of organisms using next generation sequencing (NGS). With increased sample pooling of 48 samples, Allegro V2 has been updated to enable fully automatable workflows without manual intervention. Ready-to-go automation scripts for Allegro V2 are now available on the DreamPrep NGS platform.

Using the patented Single Primer Enrichment Technology (SPET)® for DNA approach to specifically target SNPs of interest, Allegro Targeted Genotyping provides information-rich sequencing data, by capturing a SNP-specific data point for every on-target sequencing read. Coupled with DimerFree® ligation technology to eliminate adaptor dimer formation and enzymatic fragmentation for ease of use and automation, this result is unparalleled sequencing efficiency, leading to rapid scalability and the lowest cost per data point available.

Allegro Targeted Genotyping V2 kit offers several unique benefits:

  • Interrogation of over 100,000 SNPs per sample in a single assay.
  • Highly scalable multiplexing to enable largescale genotyping.
  • Reduced sequencing cost by using single end reads.
  • Complete customization to targeted SNPs, with rapid turnaround and flexibility to add new markers.
  • Automated on the DreamPrep NGS, with capablity to process 75,000 samples/year with minimal manual intervention.

Applications:

  • SNP genotyping




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Tab 02 / Highlights
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Simple, fast, robust automatable workflow

  • Simple, single-tube assay that can be completed in less than 24 hours
  • Hundreds of barcodes and increased multiplexing enables cost-saving interrogation of SNPs
  • Fully automatable on a variety of platforms

Efficient probe interrogation

  • Optimal probe placement leads to more efficient sequencing
  • Probes are designed on both sides of the SNPs within 100 bp
  • Independent probes provide validation of each SNP
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Reads from both probes confirm SNP presence

  • Zea mays custom panel designed with two probes for every SNP captures data rich information at every data point.
  • Custom targeted panels enable high accuracy SNP-based genotyping.

Optimal probe placement to capture every targeted SNP

A) Allegro Targeted Genotyping has targeted probe placement within 50 bases of the SNP.

B) SNPs are sequenced within 100 bases of every forward read, thus greatly reducing the number of sequencing cycles required.

Flexible panel designs

  • Ability to design panels for any sequenced genome
  • Our team has designed for a wide variety of over 60 species and counting
  • Expert scientists work with you to generate an optimal design approved by you

Achieve complete measurements of methylation with oxBS.

The schematic (Left) shows classic bisulfite conversion, which creates a library that detects both 5mC and 5hmC. Processing with the oxidation of 5hmC (Right) generates a bisulfite-convertible base that leads to detection of only 5mC. Differences between the libraries can then be used to deduce the sites of 5hmC modifications.

TrueMethyl oxidative bisulfite conversion enables accurate measurements of methylation.

A) Standard bisulfite conversion cannot distinguish between 5mC and 5hmC, resulting in a single readout. B) TrueMethyl oxidative bisulfite conversion provides an accurate methylation profile of each.




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Tab 03 / Specifications
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Specifications

Specification

Specification/Description

Compatible Platform

Compatible Platform

Illumina HiSeq, MiSeq, NextSeq, NovaSeq

Starting Material

Starting Material

Purified DNA

Input Amounts

Input Amounts

10 - 100 ng




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Tab 04 / Faq
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FAQ

How much DNA should I use as input? Can I use degraded DNA as input?

Allegro is designed to work with 10 to 100 ng of high quality DNA. All samples MUST be normalized prior to starting the protocol in order to obtain equal read representation after pooling. For certain small panels or large genomes, some optimization may be required; please contact technical support.

Can I pool more than 48 samples? Can I pool fewer than 48 samples?

The protocol has been optimized for hybridization of up to 48 samples of 10 to 100 ng input each. There is sufficient probe for a maximum of 4 hybridizations for each 192-reaction kit, 8 hybridizations in a 384-reaction kit, or 16 hybridizations in a 768-reaction kit.

Can I substitute my own fragmentation solution or use a mechanical/sonication method to fragment my sample?

The fragmentation cocktail has been optimized for use with downstream processing steps. Substitution with other fragmentation methods is not recommended.

Can I take the sample pools off the thermocycler to add the TX2 enzyme mix?

It is critical that the temperature is maintained at 60 °C while adding the Target Extension Enzyme Mix (TX2) to the hybridization. Mix thoroughly at this step prior to advancing to the 72 °C incubation.

Where can I stop during the protocol?

You may stop after the bead purification and/or sample concentration steps, as marked in the User Guide by a coffee cup.

Are there any special considerations when working with the adaptor plate?

Store the adaptor plate at –20 °C and keep on ice at all times, even when thawing the adaptor mixes. It is best practice to re-seal the wells after use to minimize the risk of any cross-contamination.

Can I use the standard Illumina sequencing primers with the Allegro system?

Because Allegro has been designed for the most efficient sequencing of desired targets, it requires the use of a Custom Read 1 sequencing primer. The primer is supplied at 100 μM and should be used per the manufacturer’s instructions for custom primers for the specific sequencing platform. A standard Illumina reverse sequencing primer, and standard Illumina Index 1 sequencing primer should be used. For systems with dual indexing, the use of a Custom Index 2 primer (supplied at 100 μM) will depend upon the platform and run configuration. For more information, please see User Guide section III. Planning the Experiment E. Sequencing Recommendations and Guidelines.

What is the optimal sequencer configuration for Allegro?

The system is designed to capture desired targets within a 100 base forward read. A reverse read is not required, but can be used to enhance alignment. Index 1 (and Index 2 in dual indexing configurations) is 8 bases in length.

What are your recommendations for read trimming?

See User Guide section III. G. Data Analysis Guidance of the User Guide for detailed trimming recommendations. We recommend that the first 40 nt of R1 be trimmed to remove probederived sequence. Users are advised to take precaution when using R2 for variant calling to ensure probe-derived sequence is removed. For more information, contact Tecan NGS Technical Support.




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Tab 05 / Literature
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Literature

Type
Title
No
User Guide
M01501 V1.1
Product Sheet
401002 V1
Related products
Targeted Genotyping
Targeted Genotyping

Tecan’s Allegro® Targeted Genotyping provides a fast, scalable, cost-effective approach for targeted genotyping-by-sequencing on a wide variety of organisms using NGS.

For research use only. Not for use in diagnostic procedures.