Copy-number variants in clinical genome sequencing: deployment and interpretation for rare and undiagnosed disease.

PURPOSE: Current diagnostic testing for genetic disorders involves serial use of specialized assays spanning multiple technologies. In principle, genome sequencing (GS) can detect all genomic pathogenic variant types on a single platform. Here we evaluate copy-number variant (CNV) calling as part of a clinically accredited GS test. METHODS: We performed analytical validation of CNV calling on 17 reference samples, compared the sensitivity of GS-based variants with those from a clinical microarray, and set a bound on precision using orthogonal technologies. We developed a protocol for family-based analysis of GS-based CNV calls, and deployed this across a clinical cohort of 79 rare and undiagnosed cases. RESULTS: We found that CNV calls from GS are at least as sensitive as those from microarrays, while only creating a modest increase in the number of variants interpreted (~10 CNVs per case). We identified clinically significant CNVs in 15% of the first 79 cases analyzed, all of which were confirmed by an orthogonal approach. The pipeline also enabled discovery of a uniparental disomy (UPD) and a 50% mosaic trisomy 14. Directed analysis of select CNVs enabled breakpoint level resolution of genomic rearrangements and phasing of de novo CNVs. CONCLUSION: Robust identification of CNVs by GS is possible within a clinical testing environment.

Versatile Identification of Copy Number Variants with Canvas.

Versatile and efficient variant calling tools are needed to analyze large-scale sequencing datasets. In particular, identification of copy number changes remains a challenging task due to their complexity, susceptibility to sequencing biases, variation in coverage data and dependence on genome-wide sample properties, such as tumor polyploidy, polyclonality in cancer samples, or frequency of de novo variation in germline genomes of pedigrees. The frequent need of core sequencing facilities to process samples from both normal and tumor sources favors multipurpose variant calling tools with functionality to process these diverse sets within a single software framework. This not only simplifies the overall bioinformatics workflow but also streamlines maintenance by shortening the software update cycle and requiring only limited staff training. Here we introduce Canvas, a tool for identification of copy number changes from diverse sequencing experiments including whole-genome matched tumor-normal, small pedigree, and single-sample normal resequencing, as well as whole-exome matched and unmatched tumor-normal studies. In addition to variant calling, Canvas infers genome-wide parameters such as cancer ploidy, purity, and heterogeneity. It provides fast and easy-to-run workflows that can scale to thousands of samples and can be easily incorporated into variant calling pipelines.

Canvas SPW: calling de novo copy number variants in pedigrees.

Motivation: Whole genome sequencing is becoming a diagnostics of choice for the identification of rare inherited and de novo copy number variants in families with various pediatric and late-onset genetic diseases. However, joint variant calling in pedigrees is hampered by the complexity of consensus breakpoint alignment across samples within an arbitrary pedigree structure. Results: We have developed a new tool, Canvas SPW, for the identification of inherited and de novo copy number variants from pedigree sequencing data. Canvas SPW supports a number of family structures and provides a wide range of scoring and filtering options to automate and streamline identification of de novo variants. Availability and implementation: Canvas SPW is available for download from https://github.com/Illumina/canvas. Contact: sivakhno@illumina.com. Supplementary information: Supplementary data are available at Bioinformatics online.

Canvas: versatile and scalable detection of copy number variants.

MOTIVATION: Versatile and efficient variant calling tools are needed to analyze large scale sequencing datasets. In particular, identification of copy number changes remains a challenging task due to their complexity, susceptibility to sequencing biases, variation in coverage data and dependence on genome-wide sample properties, such as tumor polyploidy or polyclonality in cancer samples. RESULTS: We have developed a new tool, Canvas, for identification of copy number changes from diverse sequencing experiments including whole-genome matched tumor-normal and single-sample normal re-sequencing, as well as whole-exome matched and unmatched tumor-normal studies. In addition to variant calling, Canvas infers genome-wide parameters such as cancer ploidy, purity and heterogeneity. It provides fast and easy-to-run workflows that can scale to thousands of samples and can be easily incorporated into variant calling pipelines. AVAILABILITY AND IMPLEMENTATION: Canvas is distributed under an open source license and can be downloaded from https://github.com/Illumina/canvas CONTACT: eroller@illumina.com SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Measurement of DNA Polymerase Incorporation Kinetics of Dye-Labeled Nucleotides Using Total Internal Reflection Fluorescence Microscopy.

We report a method for the rapid and automated measurements of the incorporation kinetics of fluorescent dye-labeled nucleotides by DNA polymerases without using stopped-flow and quench-flow methods. Total internal reflection fluorescence microscopy is used to monitor the incorporation of fluorescently labeled nucleotides by DNA polymerase into surface-bound primed DNA templates, and a microfluidic system is used to perform the reactions. We successfully demonstrated the method using Bst DNA polymerase and a set of coumarin-labeled nucleotides. Our method allows the rapid acquisition of polymerase kinetics for implementing and improving DNA sequencing technologies that rely on labeled nucleotides and DNA polymerases.

Multiplexed protein analysis using encoded antibody-conjugated microbeads.

We describe a method for multiplexed analysis of proteins using fluorescently encoded microbeads. The sensitivity of our method is comparable to the sensitivity obtained by enzyme-linked immunosorbent assay while only 5 µl sample volumes are needed. Streptavidin-coated, 1 µm beads are encoded with a combination of fluorophores at different intensity levels. As a proof of concept, we demonstrate that 27 microbead populations can be readily encoded by affinity conjugation using three intensity levels for each of three different biotinylated fluorescent dyes. Four populations of encoded microbeads are further conjugated with biotinylated capture antibodies and then combined and immobilized in a microfluidic flow cell for multiplexed protein analysis. Using four uniquely encoded microbead populations, we show that a cancer biomarker and three cytokine proteins can be analysed quantitatively in the picogram per millilitre range by fluorescence microscopy in a single assay. Our method will allow for the fabrication of high density, bead-based antibody arrays for multiplexed protein analysis using integrated microfluidic devices and automated sample processing.

Multiplexed protein detection using antibody-conjugated microbead arrays in a microfabricated electrophoretic device.

We report the development of a microfabricated electrophoretic device for assembling high-density arrays of antibody-conjugated microbeads for chip-based protein detection. The device consists of a flow cell formed between a gold-coated silicon chip with an array of microwells etched in a silicon dioxide film and a glass coverslip with a series of thin gold counter electrode lines. We have demonstrated that 0.4 and 1 µm beads conjugated with antibodies can be rapidly assembled into the microwells by applying a pulsed electric field across the chamber. By assembling step-wise a mixture of fluorescently labeled antibody-conjugated microbeads, we incorporated both spatial and fluorescence encoding strategies to demonstrate significant multiplexing capabilities. We have shown that these antibody-conjugated microbead arrays can be used to perform on-chip sandwich immunoassays to detect test antigens at concentrations as low as 40 pM (6 ng/mL). A finite element model was also developed to examine the electric field distribution within the device for different counter electrode configurations over a range of line pitches and chamber heights. This device will be useful for assembling high-density, encoded antibody arrays for multiplexed detection of proteins and other types of protein-conjugated microbeads for applications such as the analysis of protein-protein interactions.

DNA sequencing by denaturation: experimental proof of concept with an integrated fluidic device.

We report the proof of concept of a novel DNA sequencing technology called sequencing by denaturation (SBD). SBD is based on the Sanger sequencing reaction performed on amplified target templates immobilized on a solid surface followed by the denaturation of these Sanger fragments. As these fluorescently labeled fragments denature sequentially, the fluorescence intensities in the four channels corresponding to the four base types are monitored in a flow cell. A sequencing instrument with a microfluidic flowcell has been custom-designed to integrate automated fluidics, temperature control, and fluorescence imaging. The denaturation profiles of several synthetic oligonucleotides were measured with this system and our data demonstrated the ability to sequence short DNA by SBD. SBD is a simple and fast method with the potential to improve the speed and cost of large-scale genome re-sequencing.