Introduction

The reduced cost of DNA sequencing technology has led to an exponential growth of raw sequencing data. To keep pace with this development, secondary analysis tools that can provide fast and accurate results in a cost-effective manner are needed to extract actionable genomic insights. Illumina’s DRAGEN^TM (Dynamic Read Analysis for GENomics) addresses key pain points associated with next-generation sequencing (NGS) data analysis. These include a significant increase in sequencing data, limited on-premises compute options, inadequate CPU-based bioinformatics tools for rapid turnaround time, and high cost of secondary data analysis. DRAGEN empowers laboratories and organizations of all sizes to break through the bioinformatics bottleneck faster with accurate, comprehensive, and efficient secondary analysis pipelines. DRAGEN enables laboratories and researchers to do more with their genomic data by providing a robust suite of secondary analysis capabilities for a variety of experiment types, including whole genome, whole exome, RNA-Seq, methylome, and COVID detection pipelines.

DRAGEN’s DNA pipeline supports BCL to FASTQ conversion, alignment, sorting, duplicate marking, and variant calling. RNA and Methylation pipelines include RNA-Seq aligners, RNA-specific analysis, and downstream methylation calling. DRAGEN cloud offerings, powered by AWS, also include DRAGEN Apps on BaseSpace Sequence Hub^TM and DRAGEN pipelines on Illumina Connected Analytics^TM.

In this blog, we provide a walkthrough of running DRAGEN on AWS and highlight our results from the DRAGEN v4.0 DNA sequencing pipeline showing that, in addition to accuracy and efficiency, it supports the most comprehensive suite of tools for DNA sequencing, including copy number analysis, structural variants, SMN callers, repeat expansion detection, and pharmacogenomics insights for complex genes. Results from these tools are available through a single call to DRAGEN with the appropriate command line options, streamlining results for customers of all disciplines. We also evaluate the performance of the suite with respect to key metrics: runtime, cost, and concordance.

Ways to Run DRAGEN

DRAGEN’s suite of secondary analysis pipelines are offered on a variety of platforms, enabling laboratories to select a solution that best fits their needs.

DRAGEN secondary analysis pipelines are available on Illumina Connected Analytics (ICA), a secure genomics data platform built to operationalize informatics and drive scientific insights. DRAGEN on ICA empowers customers to manage, analyze, and explore large volumes of data in a secure, scalable, and collaborative environment that couples the exceptional speed and accuracy of DRAGEN.

DRAGEN Bring Your Own License (BYOL) and DRAGEN on AWS Marketplace are available for laboratories looking to run the secondary analysis platform in their existing cloud environments. DRAGEN on AWS Marketplace is available for users looking to try out DRAGEN in a flexible environment with a premium pay-as-you-go model.

Multiple options for accessing DRAGEN allow labs to select the solution that best suits the type and scale of their projects. Regular and timely updates are made to DRAGEN across the various access points to provide performance improvements, enhancements, and additional functionality. This ensures that DRAGEN’s highly accurate, comprehensive, and efficient analysis pipelines are available and up-to-date for all users.

Pre-requisites

The pre-requisites for running Illumina DRAGEN on AWS include:

• Subscription to DRAGEN on Illumina Connected Analytics, BaseSpace Sequence Hub, or run directly on AWS using DRAGEN Bio-IT Platform AMI in AWS Marketplace.

If using DRAGEN AMI in AWS Marketplace, additional pre-requisites include access to an AWS account with:

• Amazon EC2 F1 instances in public regions (North Virginia, Oregon, Dublin, or Sydney)
• Permission to use Amazon S3
• [Optional] AWS QuickStart for Illumina DRAGEN

Benchmark Analysis

The DRAGEN DNA pipeline includes optimized algorithms for mapping, sorting, duplicate marking, and haplotype variant calling. It also provides lossless compression and optimized conversion of BCL files to FASTQ format. In addition to haplotype variant calling, the pipeline supports calling of copy number and structural variants, as well as detection of repeat expansions. Figure 1 presents the different processes that are available in the DRAGEN DNA pipeline.

For benchmark analysis, we used the Genome in a Bottle (GIAB) samples HG002, HG003, and HG004 that were obtained from the Google Brain dataset available at the AWS Registry of Open Data (RODA) at s3://genomics-benchmark-datasets/google-brain/. The samples are 30x whole genomes aligned to the hg38 alt-masked graph v2 genome hash table from Illumina.

We ran the DNA pipeline of DRAGEN v4.0 software using FASTQ files as input to perform mapping/alignment and calling/genotyping of all variants that the pipeline is capable of, including small variant, structural variant, CYP2B6, CYP2D6, GBA, SMN, PGx Star Allele, and repeat expansion detection with an expanded catalog of Short Tandem Repeat (STR) loci available beginning with v4.0. Mapping/alignment and comprehensive variant calling were performed using a single command line that runs all the pipelines in an end-to-end workflow, thereby eliminating the need for manual step-wise processing by the user. For comparative analysis, we ran the experiment on the three samples across f1.4xlarge and f1.16xlarge EC2 instances.

Launch F1 instance with DRAGEN AMI

DRAGEN is available on AWS Marketplace as an Amazon Machine Image (AMI). An AMI provides the information and pre-requisites to launch an EC2 instance. After subscribing to the DRAGEN AMI, we launched two EC2 F1 instances: f1.4xlarge and f1.16xlarge with 500 GB root volume for storage and default VPC and subnet.

As an alternative to launching the F1 instance using the DRAGEN AMI directly via SSH, customers can also use the Illumina DRAGEN QuickStart from AWS, which deploys the necessary backend infrastructure in the customer’s account, with minimal effort from the customer. The DRAGEN QuickStart, which leverages AWS Batch, allows customers to simplify the processing of 100’s or 1000’s of samples by submitting them to a job queue. AWS Batch manages the execution of the jobs, so that the customer does not need to manage any compute clusters.

Run DRAGEN DNA Pipeline

We executed the following commands to run DRAGEN’s end-to-end alignment and variant calling pipeline. This takes the paired-end compressed FASTQ files and reference hash table as input and generates a map/aligned, sorted, and duplicate-marked CRAM file and variant calls in VCF and gVCF file formats as output. We enabled flagging of duplicate output alignment records, saving of the output from the map/align stage, small variant calling, structural variant (SV) calling, copy number variant (CNV) calling in self normalization mode, CYP2D6 diplotyping, filtering of variant calls, and repeat genotyping (or expansion detection) using the corresponding flags: enable-duplicate-marking, enable-map-align-output, enable-variant-caller, enable-sv, enable-cnv, cnv-enable-self-normalization, enable-cyp2d6, vc-hard-filter, repeat-genotype-enable, respectively. Although we tested DRAGEN with BYOL option, customers can also use DRAGEN from AWS Marketplace to run these experiments.

The following is the DRAGEN command line used…

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