VCAT v4.0 – Support for DRAGEN and GA4GH best practices for small variant benchmarking

Author: Eric Allen, Associate Director of Bioinformatics at Illumina

The Variant Calling Assessment Tool (VCAT) v4.0 BaseSpace™ App has an updated hap.py and several new gold references and panel BED files.

This update includes a recent version of hap.py which (in combination with vcfeval) has been selected by the GA4GH as the recommended tool for small variant call benchmarking. For more details, see the publication at: https://www.ncbi.nlm.nih.gov/pubmed/30858580

Updated and improved tools

Gold Reference Additions and Updates

  • Updated Platinum Genomes reference data to version 2017-1.0
  • Added all the NIST GiaB gold references to v3.3.2:
    • Caucasian female: NA12878 (mother)
    • Ashkenazi Jewish trio: NA24143 (mother), NA24149 (father), NA24385 (son)
    • Han Chinese trio: NA24695 (mother), NA24694 (father), NA24631 (son)

More built-in panel BED files

  • Illumina, IDT, and Twist exome panels
  • Several AmpliSeq panels

The diagram below shows how VCAT, hap.py, and vcfeval work together.

See the screenshots below displaying some of the new features.

We hope you find VCAT 4.0 useful. Questions can be directed to basespacelabs@illumina.com.

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

QB#9047

MiXCR Immune Repertoire Analyzer version 2.1.11 from MiLaboratories is now available at Illumina BaseSpace™

  • Youting Sun, Senior Bioinformatics Scientist at Illumina
  • Dmitriy Chudakov, CSO at MiLaboratories https://milaboratory.com

The upgraded version of the MiLaboratories LLC flagship software product, MiXCR, is now officially available as an Illumina BaseSpace™ application.

MiXCR is a “gold standard” analytical package in the area of T-cell receptors (TCR) and immunoglobulin (IG) repertoire profiling. Analysts apply MiXCR for extracting immune repertoires  from any type of sequencing data with any level of TCR/IG coverage, ranging from perfectly enriched libraries such as multiplex PCR or targeted 5’RACE, to the “rare event” datasets containing several target entities among hundreds of millions of reads, such as RNA-Seq, and even Exome-Seq data.

In the new version, simple selection of species (Human or Mouse), template material (RNA or DNA), library type (targeted or random), and basic information on library preparation enables appropriate analysis settings for a variety of immune repertoire experiment scenarios:

MiXCR Immune Repertoire Analyzer v2.1.11 Input Form Parameters.

Extraction of both full VDJ length or CDR3  only repertoires is possible, for TCR or IG chains of interest, with or without out-of-frame and stop codon-containing clonotype variants:

MiXCR Immune Repertoire Analyzer v2.1.11 Analysis Settings.

The application also provides post-analysis metrics in the form of interactive reports, including:

Post-analysis metrics in the form of interactive reports, including basic statistics.
Spectratype with major clonotypes.
Quantile Statistics on clonotype frequencies.
Clonotypes with colorized V, D, and J segments.

High extraction efficiency for any type of sequencing data and superb accuracy should make the new MiXCR BaseSpace version a highly useful resource for many. The ability to look at basic parameters and immediately download the resulting figures for reports and publication make it really convenient for the most efficient everyday work on immune repertoires.

Analysis of amplicon data

Recommended settings for panels (a) Immune repertoire panel and (b) TCR-beta SR panel:

  • Starting material: RNA for panel (a), RNA or Genomic DNA for panel (b)
  • Library type: Targeted TCR/IG library amplification (5’RACE, Amplicon, Multiplex, etc)
  • 5’-end of the library: V gene single primer/multiplex
  • 3’-end of the library: J gene single primer/multiplex
  • Presence of PCR primers and/or adapter sequence: Absent / nearly absent / trimmed
  • Target region: CDR3

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

QB#9017

DRAGEN™ Enrichment App – Accurate, rapid analysis for germline and somatic exome experiments

Author: Eric Allen, Associate Director of Bioinformatics at Illumina

As part of the new DRAGEN v3.4 launch, the Illumina software development team has released a new BaseSpace-exclusive DRAGEN app –DRAGEN Enrichment v3.4. Combining the best of DRAGEN with Illumina’s legacy Enrichment 3 App, the DRAGEN Enrichment app provides ultra-rapid analysis and improved accuracy all at a lower cost per sample.

The DRAGEN Enrichment app is the preferable method for analyzing enrichment data with DRAGEN, delivering a full suite of enrichment specific metrics and reporting.

Here is what to know:

  • The DRAGEN Enrichment App is faster and more accurate vs Enrichment (Isaac/Starling) and BWA Enrichment (BWA/GATK) apps, as demonstrated via the visuals below
  • Variant Calling:
    • Small variant calling – The app includes germline and somatic (low-frequency) small variant calling (tumor only); outputs VCF and gVCF in same analysis
      • Note: Tumor-normal analysis can be conducted by first running the DRAGEN Enrichment app on all their normal and tumor samples, and then running the DRAGEN Somatic app on the resulting BAM files for the Tumor/Normal pairs.
    • Copy number variant (CNV) calling – utilize CNV baseline files based on a panel of normals
    • Structural variant calling
  • Enrichment metrics generated:
    • Read/base enrichment padded/unpadded
    • Uniformity
    • % bases covered at 1x, 10x, 20x, 50x
    • Picard HsMetrics enabled by checkbox
  • Variety of reference options supported, including hg19, GRCh38 and custom references
  • Includes built-in targeted region BED files for common enrichment panels, and accepts custom targeted region BEDs
  • Extensive reporting:
    • In-browser, PDFs, and CSVs
    • Single sample and aggregate reports
  • Integrated variant annotation (Nirvana) and variant browser

The improved small variant calling over other available BaseSpace app solutions is shown below for one replicate of Coriell sample NA12878 with 106x depth:

Analysis AppApp Execution TimeDRAGEN-only Execution TimeSNV RecallSNV PrecisionIndel RecallIndel Precision
DRAGEN Enrichment v3.4.516m 4s6m 50s95.04%99.49%86.90% 92.18%
(Isaac/Starling) Enrichment v3.1.053m 20sNA93.26%99.38%78.29% 86.90%
BWA Enrichment v2.1.21h 23m 2sNA90.66% 99.78%72.85% 89.44%


• Example sample (s01-NFE-CEX-NA12878-demo.vcf) was prepared using Nextera Flex for Enrichment Library Preparation kit with dual indices and sequenced on a NovaSeq™ S2 flow cell: https://basespace.illumina.com/s/FaxWSm2X1gwO
• Variant accuracy comparison was performed using the Variant Calling Assessment Tool v3.2.0 app.

CNV calling is also enabled in the DRAGEN Enrichment app. The screenshot below from IGV shows a 937,697 bp CNV loss found in a melanoma cancer sample (Me01/ERR174231) around the chromosomal region chr9:125239269-126176965. The sample data was obtained from NCBI’s Sequence Read Archive (accession ERR174231) using the SRA Import BaseSpace App.

Project: SRA: ERP001844 (Agilent SureSelect – Exome CNV Detection – Melanoma). Publication: Magi et al.

Somatic/low-frequency variant calling is also enabled. The table below demonstrates the usefulness of this somatic calling tool:

Variant TypeChr Pos Gene Variant HD753 – Expected VF (%) HD753 – Measured VF (DRAGEN Enrichment) (%)
SNV Low GCchr.3 178936091 PIK3CA E545K 5.63.8
SNV High GC chr.19 3118942 GNA11 Q209L 5.66
Long Deletion chr.7 55242464 EGFR ΔE746 – A750 5.33.3
Long Insertion chr.755248998 EGFR V769_D770insASV 5.63.7
SNV High GC chr.14 105246551 AKT1 E17K 55.7

Project: NovaSeq S4: Nextera Flex for Enrichment (HCC1187, HCC1395, HCC1954, HD753, Coriell Mixture). 1% VF cutoff

We’ve also incorporated many of the comprehensive metrics and reporting features built into the legacy Enrichment 3.1.0 app, including read-, base-, and target-level enrichment metrics, as well as the variant table for simple variant call browsing and filtering.

We hope this update enables you to discover new insights. Stay tuned for more app announcements, and let us know if you have any questions.

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
QB#8389

Doing more with DRAGEN™ v3.2.8

Advancing Workflows through Relentless Innovation

We’ve been busy over the last few months! Back in May, Illumina announced the acquisition of Edico Genome and the DRAGEN™ (Dynamic Read Analysis for GENomics) technology. Since then, we have been hard at work expanding DRAGEN’s capabilities to provide more advanced, robust and performant pipelines for our customers. With the inclusion of DRAGEN into the Illumina ecosystem, we are now able to take advantage of the expertise of both teams to build out an expanded chest of tools that offer added functionality, benefits and ease-of-use.

The team has come a long way since we last published about DRAGEN on the BaseSpace™ Blog, and we are excited to share some insight into what we have been working on. Over the coming months, we will continue to post about our latest updates and activities to keep you updated.

Earlier this month, we released DRAGEN v3.2.8, which introduces a variety of new capabilities designed to deliver more insights from your data.

Continue reading Doing more with DRAGEN™ v3.2.8

Join us for our upcoming webinar

Join us for our upcoming webinar: High-volume sequence analysis with BaseSpace Sequence Hub and Edico DRAGEN apps, on Dec 13 at 10AM (PT)

The latest sequencing technologies enable unprecedented throughput and redefine limits for many labs. To adapt, these labs must redefine how they work – by automating tasks to reduce touchpoints and by simplifying workflows with integration and robust analysis tools.

In this webinar, we describe BaseSpaceSequence Hub and how the newest features support high throughput, high-volume sequencing. We demonstrate how customers can progress from flowcell loading to variant analysis with zero touchpoints by using the Whole Genome Sequencing or Edico DRAGEN apps. Additionally, we describe how the integration with BaseSpace Variant Interpreter enables users to interpret and generate reports of identified variants.

Register now

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

 

Hap.py app on BaseSpace® Sequence Hub: GA4GH benchmarking of small variant calls

Bioinfomatics tools are a key component in the Next-generation Sequencing (NGS) workflow and can have a significant impact on the results. Alignment and variant calling, in particular, involve complex algorithms, each with unique strengths and weaknesses. The Broad Institute’s BWA+GATK application is among the most popular, but over the last few years more alignment+variant calling methods have been released by companies including Illumina, Edico Genome, and Sentieon. With the emergence of multiple methods comes a clear need for comparison between the results obtained by these methods so that people who use these tools can select the best one for their purpose.

The new Hap.py app available on BaseSpace Sequence Hub enables users to compare diploid genotypes at the haplotype level by generating and matching alternate sequences in a small region of the genome that contains one or more variants. Hap.py makes it easy to compare any variant call set against a range of packaged gold-standard truth sets1,2 to perform routine benchmarking.

Continue reading Hap.py app on BaseSpace® Sequence Hub: GA4GH benchmarking of small variant calls

Edico Genome’s DRAGEN Bio-IT Platform Now Available on BaseSpace® Sequence Hub

Removing the NGS Analytics Data Bottleneck with Field-Programmable Gate Arrays (FPGAs)

The following is a guest blog, written by our partners at Edico Genome.

The next-generation sequencing (NGS) analysis demand is growing at an exponential rate, creating a shortage of computing power to analyze the rapidly growing body of data. Current projections1 calculate genomic data to continue doubling every seven months, a stark acceleration in comparison to Moore’s Law, which states CPU capabilities will double every two years (Figure 1, below). The void left in-between creates a bottleneck for genomics labs.

Picture1
Figure 1

Providing an alternative to traditional CPU-based systems, Edico Genome’s DRAGEN™ (Dynamic Read Analysis for Genomics) Platform leverages FPGA (Field-Programmable Gate Array) technology to provide customers with hardware-accelerated implementation of genome pipeline algorithms. Leveraging FPGAs, DRAGEN allows customers to analyze NGS data at unprecedented speeds with extremely high accuracy2 onsite, in the cloud, or through a blended hybrid cloud.

BaseSpace Sequence Hub, hosted on Amazon Web Services, enables the cloud-based deployment of the Edico Genome DRAGEN pipeline. Edico Genome’s DRAGEN Genome Pipeline is now readily available, enabling rapid analysis of whole genome sequencing and targeted resequencing panels.

Continue reading Edico Genome’s DRAGEN Bio-IT Platform Now Available on BaseSpace® Sequence Hub