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Focus On Your Target

Grace DeSantis, Tatjana Singer, and Gary P. Schroth. Illumina Applications Group
| Oct 07, 2015

Here at Illumina, we take immense pride in creating accessible, versatile sequencing products and software tools. Our Applications Group specializes in actively pushing the limits and improving upon our existing technologies with a focus on targeted sequencing applications. Yet, we recognize that sequencing is not an end in itself, but rather a tool for discovery. Challenges are now less about how to sequence and more about interpretation, innovation, and insight. We seek to simplify the NGS workflow and increase quality and performance to free up your time to focus on the questions and challenges of the next research project.

Today, we announced the launch of the TruSeq® Custom Amplicon (TSCA) Low Input Library Preparation kit with some important updates to DesignStudio, to complement the TruSeq Amplicon Analysis App on BaseSpace. Allowing very low sample input from formalin-fixed, paraffin embedded (FFPE) DNA samples, TSCA Low Input enables sensitive variant frequency detection. The fully customizable assay allows high amplicon multiplexing (up to 1536 amplicons and a range of amplicon sizes), giving consistent performance across sample inputs, allowing you to confidently call variants down to 5%. The end-to-end assay workflow allows translational researchers to focus on getting the results that matter, particularly from challenging samples, with minimal effort, time, and resources.

Figure 1 (click to enlarge):
The TSCA Low Input Library Prep Kit is part of an integrated, fully-supported workflow for amplicon sequencing that guides you from design through data analysis.

Simplify Custom Design

DesignStudio™ is a web-based assay design tool that allows researchers to create custom amplicon designs to fit your research interests and study needs using a step-by-step tool with a new streamlined interface. DesignStudio provides dynamic feedback to optimize target region sequencing coverage, reducing the overall design time. At the push of a button, your custom designs are synthesized, paired with the appropriate kit biochemistry and shipped to you. New DesignStudio updates include design support for many species including rice, soybean, maize, pig, sheep, chicken, and canine.

Detect with Confidence: Dual Strand Sequencing

The TruSeq Custom Amplicon solution offers the unique option of dual strand sequencing. The dual strand extension-ligation approach using a high fidelity polymerase allows independent and accurate interrogation of both DNA strands to distinguish deamination artifacts from true mutations (Figure 2A and 2B). Since FFPE artifacts typically affect only one of the base pairs, this approach offers more power than simply doing replicate testing.

Figure 2 (click to enlarge): Optional dual strand sequencing, now enabled in Design Studio, allows easy differentiation of damage artifacts from FFPE treatment (A) from true somatic mutations (B).

Consistent Variant Detection

A common struggle with targeted sequencing experiments is the desire to achieve sensitive variant frequency detection despite having very low amounts of sample. TSCA Low Input requires only 10-50 ng FFPE DNA (depending on quality), and is sensitive enough to accept as low as 1 ng of input for high quality DNA samples (Figure 3A and 3B). The consistent assay performance across a range of DNA inputs inspires confidence in your results with call variants of >5%, and in some cases as low as 2–3 % (Figure 4A and 4B). 


Figure 3 (click to enlarge):
Mutation detection correlation plots demonstrating technical reproducibility of TSCA Low Input Library Preparation with control sample at various inputs (1, 5, and 10 ng). A. Robust detection of SNVs in sample HD200 ranging from 24.5% to 2.5% frequency with input from 1-10 ng. B. Robust detection of SNVs in sample HD749 ranging 30% to 4% frequency with input range of 10 ng. 


Figure 4: (click to enlarge) A. Robust specificity and uniformity achieved with highly degraded FFPE samples derived from three tissues: stomach (St), rectal (Re),and lung (Lu) matched tumor (C) and normal (N). B. Robust mutation detection and low false positive rates in a FFPE tumor/normal matched stomach sample (KRASG13D). This browser shot shows two replicates with 10, 20, and 50 ng DNA input, with all of the calls above 5% variant frequency.

QC, Analysis, and More with BaseSpace

For those new to sequencing technologies, the BaseSpace analysis portal is a great way to get started and explore your data without having to make major investments in computer hardware or learning command-line software. MiSeq® Reporter and BaseSpace take all the guesswork out of sequencing analysis. If you are an advanced user, you’ll appreciate the BaseSpace cloud storage solutions and easy tools for first-pass analyses and quality control before expending your own compute resources. Experienced users may have configured their own storage systems and sophisticated analysis pipelines. If you already have a great pipeline you’d like to share, we’re hoping that you’ll become a BaseSpace developer, and enable other Illumina customers to use the great tools that you've developed.

New upgrades to BaseSpace Amplicon Apps are coming soon, including the ability to adjust variant threshold to suite your project needs, with a 5% variant threshold default. Get started with analysis by checking out an example TruSeq Custom Amplicon Low Input data set on BaseSpace (free login required). This data set consists of several libraries generated from high quality human genome samples including 2800M, commercial FFPE reference materials HD200, HD-C749, and HD729 as well as human FFPE tumor/normal samples prepared using TSCA Low Input biochemistry and sequenced on a MiSeq system. The library prep chemistry has also been validated on the NextSeq system.

Life appears to move faster with each passing day. We strive to do more science, to discover more, and to have more purposeful time pursuing our passions, on and off the bench. With a smart and seamless workflow solution for amplicon sequencing, the TruSeq Custom Amplicon Low Input kit enables researchers to process even the most challenging samples confidently. Our sample-to-answer solutions are designed to save time for your ideas to come to fruition, and to be ready for the next challenge. And you’re not alone; Illumina’s highly-trained technical and field specialists, as well as the combined expertise of our support team, ensure rapid resolution and minimize potential laboratory downtime. We can’t make time stand still, but we can help make your work more efficient and effective.

For Research Use Only.  Not for use in diagnostic procedures.